Biogenesis and Metabolic Maintenance of Rubisco

PP68CH02-Hayer-Hartl ARI 6 April 2017 10:12

Biogenesis and MetabolicMaintenance of Rubisco∗

Andreas Bracher,1 Spencer M. Whitney,2

F. Ulrich Hartl,1 and Manajit Hayer-Hartl11Department of Cellular Biochemistry, Max Planck Institute of Biochemistry,82152 Martinsried, Germany; email: [email protected], [email protected],[email protected] School of Biology, Australian National University, Acton, Australian CapitalTerritory 2601, Australia; email: [email protected]

Annu. Rev. Plant Biol. 2017. 68:29–60

First published online as a Review in Advance onJanuary 11, 2017

The Annual Review of Plant Biology is online atplant.annualreviews.org

https://doi.org/10.1146/annurev-arplant-043015-111633

∗ c© Crown copyright 2017. Reproduced with thepermission of the Controller of Her Majesty’sStationery Office/Queen’s Printer for Scotlandand the Research School of Biology, AustralianNational University, Canberra.

Keywords

Rubisco, assembly chaperone, chaperonin, Rubisco activase, metabolicrepair

Abstract

Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) mediates thefixation of atmospheric CO2 in photosynthesis by catalyzing the carboxy-lation of the 5-carbon sugar ribulose-1,5-bisphosphate (RuBP). Rubisco isa remarkably inefficient enzyme, fixing only 2–10 CO2 molecules per sec-ond. Efforts to increase crop yields by bioengineering Rubisco remain un-successful, owing in part to the complex cellular machinery required forRubisco biogenesis and metabolic maintenance. The large subunit of Ru-bisco requires the chaperonin system for folding, and recent studies haveshown that assembly of hexadecameric Rubisco is mediated by specific as-sembly chaperones. Moreover, Rubisco function can be inhibited by a rangeof sugar-phosphate ligands, including RuBP. Metabolic repair depends onremodeling of Rubisco by the ATP-dependent Rubisco activase and hydroly-sis of inhibitory sugar phosphates by specific phosphatases. Here, we reviewour present understanding of the structure and function of these auxiliaryfactors and their utilization in efforts to engineer more catalytically efficientRubisco enzymes.

29

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ANNUAL REVIEWS Further

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http://www.annualreviews.org/doi/full/10.1146/annurev-arplant-043015-111633


Rubisco: ribulose-1,5-bisphosphatecarboxylase/oxygenase

RuBP: ribulose-1,5-bisphosphate

Contents

INTRODUCTION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30RUBISCO STRUCTURE AND FUNCTION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32

Structure of Large and Small Subunits . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32Reactions Catalyzed by Rubisco . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35

RUBISCO SUBUNIT FOLDING . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36The Bacterial Chaperonin. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38The Chloroplast Chaperonin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38

CHAPERONES FOR RUBISCO ASSEMBLY. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40RbcX . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41Raf1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43Raf2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44Red-Type RbcS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45The Challenge of Plant Rubisco Reconstitution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46

METABOLIC MAINTENANCE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46Prokaryotic Rca . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48Eukaryotic Rca . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49Inhibitory Sugar Phosphatases. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49

PERSPECTIVES ON RUBISCO ENGINEERING . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50Evolutionary Considerations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50Strategies to Enhance Rubisco Function . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53

OUTLOOK . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53

INTRODUCTION

Life on earth depends on the ability of photosynthetic organisms to sequester inorganic CO2

from the atmosphere into organic carbon compounds via the Calvin-Benson-Bassham cycle ofphotosynthesis (Figure 1). A key enzyme in this process is ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco), the most abundant protein in nature (26). The amount of CO2 fixed byRubisco is estimated at 250 billion tons per year (36). Recent forecasts have suggested that globalfood production will need to rise more than 30% by 2050 to meet the demands of an ever-increasinghuman population (31, 71). Because all biomass (and thus all food sources) results either directlyor indirectly from the action of Rubisco in photosynthesis, improving crop yields will requirestrategies to increase the efficiency of carbon fixation. Reengineering a more efficient Rubiscoenzyme is central to this approach, especially when considering that adaptation of crop plants toclimate change might be necessary to avoid a bottleneck in food production (57, 87, 90, 103, 140).

Photosynthesis occurs in plants, algae, and many species of bacteria, with the light-dependentreactions taking place on the thylakoid membranes of chloroplasts in plants and algae andon plasma membrane invaginations (chromatophores) in bacteria (48). The light reactionsconvert energy from sunlight into chemical energy in the form of ATP and reducing equivalents(NADPH), which are used in the light-independent Calvin-Benson-Bassham cycle in thechloroplast stroma to produce carbon compounds and regenerate the 5-carbon sugar substrate ofRubisco, ribulose-1,5-bisphosphate (RuBP) (Figure 1). Rubisco catalyzes the key step of carbonfixation by carboxylating RuBP to produce an unstable 6-carbon intermediate and processingit into two molecules of 3-phosphoglycerate (3PGA). The 3PGA is then converted in the

30 Bracher et al.

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RubiscoCO2

G3P

NADPHATP

Sugars

RuBP

3PGA

2PG

Photosyntheticcarbon fixation

(CBB cycle)

Chloroplast

Peroxisome

Mitochondrion

Photorespiration

NADPADP

Thylakoids

Photosyntheticlight reactions

Light

e–, H+

Plant growth

O2

CO2

O2

H2O

ATP, NADPH

Figure 1Schematic view of the role of Rubisco in photosynthesis. The light reactions provide energy and reducingagents (ATP and NADPH), which are used in the light-independent CBB cycle to produce carboncompounds (sugars). In the CBB cycle, Rubisco catalyzes the key step of fixation of atmospheric CO2 bymediating the carboxylation of the 5-carbon sugar substrate RuBP. The resulting 3PGA molecules are thenconverted to G3P via a series of steps, giving rise to the synthesis of sugars, and are also used to regenerateRuBP. Rubisco can also accept oxygen as a substrate instead of CO2, resulting in the formation of the toxicproduct 2PG. Recycling of 2PG back into 3PGA by photorespiration comprises a complex enzymaticpathway involving the transport of metabolites between chloroplasts, leaf peroxisomes, and mitochondria,requiring additional energy, and also results in loss of fixed CO2. Abbreviations: CBB, Calvin-Benson-Bassham; G3P, glyceraldehyde-3-phosphate; 2PG, 2-phosphoglycolate; 3PGA, 3-phosphoglycerate;Rubisco, ribulose-1,5-bisphosphate carboxylase/oxygenase; RuBP, ribulose-1,5-bisphosphate. Figuremodified from Reference 109 with permission from Elsevier.

Calvin-Benson-Bassham cycle to glyceraldehyde-3-phosphate (G3P) via a series of steps thatrequire ATP and NADPH. G3P gives rise to the synthesis of sugars, fatty acids, and amino acidsand is also used to regenerate RuBP (103) (Figure 1).

Considering its pivotal role, Rubisco is surprisingly inefficient as an enzyme. Its slow catalyticrate of only ∼2–5 CO2 molecules fixed per second in plants necessitates the production of sig-nificant levels of Rubisco, which can constitute as much as 50% of the soluble leaf protein (35).Moreover, as the name indicates, Rubisco can also accept oxygen as a substrate instead of CO2. Itis important to note in this context that Rubisco evolved more than 3.5 billion years ago in an at-mosphere free of oxygen and rich in CO2 (82, 140). The competing reaction of RuBP with oxygenproduces one molecule of 3PGA and one molecule of 2-phosphoglycolate (2PG), which is toxic tochloroplasts (148). Recycling of the 2PG back into 3PGA for use in the Calvin-Benson-Basshamcycle occurs via photorespiration, a complex enzymatic pathway involving transport of metabo-lites between chloroplasts, peroxisomes, and mitochondria (135) (Figure 1). Some organismslargely circumvent the energy expense and loss of CO2 during photorespiration by concentratingCO2 around Rubisco (76). Disruption of photorespiration in plants that lack CO2-concentratingmechanisms perturbs carbon and nitrogen cycling, indicating that this pathway is physiologi-cally relevant (13, 135). As discussed below, another complication of Rubisco catalysis is that the

www.annualreviews.org • Rubisco Biogenesis and Repair 31

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Folding: the processby which an extendednewly synthesizedpolypeptide chaincollapses into itsfunctionalthree-dimensionalconformation

Assembly: theassociation of two ormore proteinmolecules to afunctional complex

Chaperone: a proteinthat binds to andstabilizes an otherwiseunstable conformer ofanother protein andfacilitates its correctfate in vivo

RbcL: Rubisco largesubunit

RbcS: Rubisco smallsubunit

multistep reactions of both carboxylation and oxygenation are error prone, producing sugar phos-phate by-products that bind tightly to the active site and inhibit Rubisco function (45, 91).

These shortcomings have provided a strong incentive for efforts to engineer a better Rubiscoenzyme. Although it has been possible to enhance the overall catalytic performance of bacterialand archaeal Rubisco (25, 143), there has been no success in improving plant Rubisco (90). Thismight be due, at least in part, to constraints imposed by the complex cellular machineries requiredfor the folding, assembly, and metabolic maintenance of Rubisco. Here, we review recent advancesin understanding the role of auxiliary factors in Rubisco biogenesis and metabolic repair. We beginby summarizing the structure and function of Rubisco, and then describe the chaperone systemsthat mediate the folding and assembly of Rubisco subunits. We then review the Rubisco activasesand specific phosphatases that reverse Rubisco autoinhibition. Finally, we discuss past and presentapproaches used to engineer Rubisco enzymes with improved catalytic properties.

RUBISCO STRUCTURE AND FUNCTION

Phylogenetic analyses indicate the existence of three classes of bona fide Rubisco proteins, referredto as forms I, II, and III (121). The most abundant is form I, an∼550-kDa hexadecameric complexconsisting of eight large (RbcL,∼50–55 kDa) and eight small subunits (RbcS,∼12–18 kDa), whichoccurs in plants, algae, cyanobacteria, and proteobacteria (3). Form I Rubiscos are further classifiedinto green-type (in proteobacteria, cyanobacteria, green algae, and plants) and red-type enzymes(in photosynthetic bacteria and nongreen algae) (7, 119, 121) (Figure 2). The core of the complexcomprises a tetramer of antiparallel RbcL dimers carrying the active sites, with four RbcS subunitsat the top and four at the bottom (Figure 3a,b). The shape of the oligomer resembles a cylinderwith a diameter of ∼110 A and a height of 100 A. The simpler form II Rubiscos, which occur inbacteria and dinoflagellates, lack RbcS and comprise one or more RbcL dimers. Form III Rubiscosare found in archaea and are ring-shaped assemblies of three to five RbcL dimers. In contrast toform I and II Rubiscos, form III Rubiscos serve in biological roles other than photosyntheticfunctions, such as the regeneration of RuBP produced during nucleotide metabolism (120, 121).Similarly, the so-called Rubisco-like proteins in some bacteria, archaea, and algae do not catalyzeRuBP carboxylation or oxygenation (121). In eukaryotes, the RbcL subunits are always encoded bythe chloroplast genome (plastome), whereas the RbcS subunits are encoded in the plastome onlyin nongreen algae and are encoded in the nucleus in plants and green algae (119). Cyanobacteriaconcentrate Rubisco in proteinaceous compartments called carboxysomes (101), whereas greenalgae, such as Chlamydomonas reinhardtii, package it in chloroplast-associated microcompartmentscalled pyrenoids (136). These compartments contain additional machinery (proton/carbonatepumps and carbonic anhydrase) to generate locally high CO2 concentrations around Rubisco (30,32, 55, 102).

Structure of Large and Small Subunits

Crystallographic studies show high structural similarity of the RbcL subunits in the various formsof Rubisco (3), consistent with the high sequence identity among species (∼60% in form I Rubisco)(Figure 3a,b). RbcL consists of an N-terminal α + β domain of ∼150 residues and a C-terminaldomain of ∼325 residues, which contains a β8α8 triose-phosphate isomerase (TIM)–barrel do-main of∼310 residues and a flexible C-terminal tail (defined as all residues from Trp462, tobacconumbering, to the C terminus) of ∼15 residues. The antiparallel RbcL dimer has two active siteslocated at the interface between the N-terminal domain of one subunit and the C-terminal TIM-barrel domain of the adjacent subunit (Figure 3c). To become catalytically competent, Lys201

32 Bracher et al.

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H. neapolitanus

T. ferrooxidans

N. agilis

P. marinus

Syn. WH7803

Syn. PCC6301

Syn. PCC7002Syn. PCC6803Fischerella sp.

JSC-11

N. azollae

Nostoc sp.

PCC7120

T. vulcanus

C. re

inha

rdtii

P. p

aten

s

P. sa

tivumG.

max

G. hirs

utum

A. thalia

naN. tabacum

S. oleracea

Z. mays

Cylindrotheca N1

D. baltica

P. tricornutum

O. sinensis

T. oceanica

T. pseudonanaO. luteus

G. monilis

P. purpurea

G. p

artit

a

G. s

ulph

urar

ia

R. eu

troph

aR.

spha

eroi

des

X. flavus

Form ICFolding: GroEL-GroESAssembly: RbcSRbcL C terminus: WGXISFN(S/P)TDXXD(F/Y)XXXXXXXActivation: Prokaryotic Rca (red type)

Form IAFolding: GroEL-GroESAssembly: Raf2RbcL C terminus: WKEIKFEF(D/E)TXDKLXXXXActivation: Prokaryotic Rca + adapter (CbbQ-CbbO)

Form IB (prokaryote)Folding: GroEL-GroESAssembly: RbcX, Raf1RbcL C terminus: WKEIKFEFXXXDXXActivation: ?

Form IB (eukaryote)Folding: Cpn60/Cpn20/Cpn10 Bsd2Assembly: RbcX, Raf1, Raf2RbcL C terminus: WKEIXFXFXXXDXXActivation: Eukaryotic Rca

Form IDFolding: Cpn60/Cpn20/Cpn10Assembly: ?RbcL C terminus: WKDI(S/T)FNYTSTDXXD(F/Y)XXXXXXXActivation: Eukaryotic Rca (red type)

GREEN-TYPE ENZYMES

RED-TYPE ENZYMES

Figure 2Molecular phylogenetic tree of selected Rubisco RbcL sequences. The green-type enzymes encompass forms IA and IB, and thered-type enzymes encompass forms IC and ID. Components involved in Rubisco folding, assembly, and activation identified in thedifferent clades are indicated. The RbcL C-terminal sequences are important for Rubisco assembly and functional maintenance.Variable residues are indicated by X. The phylogenetic tree was calculated with T-Coffee (84) by multisequence alignment, and thediagram was created using the software Dendroscope (49). Abbreviations: Bsd2, bundle sheath defective 2; Rca, Rubisco activase; Raf,Rubisco accumulation factor; Rubisco, ribulose-1,5-bisphosphate carboxylase/oxygenase. Species names (counterclockwise from middleleft): Z. mays, Zea mays; S. oleracea, Spinacia oleracea; N. tabacum, Nicotiana tabacum; A. thaliana, Arabidopsis thaliana; G. hirsutum,Gossypium hirsutum; G. max, Glycine max; P. sativum, Pisum sativum; P. patens, Physcomitrella patens; C. reinhardtii, Chlamydomonasreinhardtii; T. vulcanus, Thermosynechococcus vulcanus; N. azollae, Nostoc azollae; Syn. PCC6803, Synechocystis PCC6803; Syn. PCC7002,Synechococcus PCC7002; Syn. PCC6301, Synechococcus PCC6301; Syn. WH7803, Synechococcus WH7803; P. marinus, Prochlorococcusmarinus; N. agilis, Nitrobacter agilis; T. ferrooxidans, Thiobacillus ferrooxidans; H. neopolitanus, Halothiobacillus neopolitanus; X. flavus,Xanthobacter flavus; R. sphaeroides, Rhodobacter sphaeroides; R. eutropha, Ralstonia eutropha; G. sulphuraria, Galdieria sulphuraria; G. partita,Galdieria partita; P. purpurea, Porphyra purpurea; G. monilis, Griffithsia monilis; O. luteus, Olisthodiscus luteus; T. pseudonana, Thalassiosirapseudonana; T. oceanica, Thalassiosira oceanica; O. sinensis, Odontella sinensis; P. tricornutum, Phaeodactylum tricornutum; D. baltica, Durinskiabaltica. Figure modified from References 45 and 121.


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(plant numbering) in the active-site pocket is covalently modified by carboxylation, forming a car-bamyl group that subsequently binds a Mg2+ ion. This modification, referred to as carbamylation,is necessary for correct RuBP binding in the active site. Upon binding of RuBP, the mobile loop6 region of the TIM barrel and the flexible C-terminal RbcL sequence form a multilayered lid

a

e

RbcS

ABEF

N

N-terminaldomain

c d

Loop 6CABP

C-terminaldomain

RbcL2 unit

N

Side view Top viewGreen-type RbcL8S8

Green-type RbcS Red-type RbcS Superposition

b Side view(cross section)

Top viewRed-type RbcL8S8

RbcS

RbcL

RbcL

AB

EFAB

100 Å

110 Å

Mg

C

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XuBP: D-xylulose-1,5-bisphosphate

that closes the active site and generates the physical environment required for electrophilic attackof RuBP by CO2 or O2 (Figure 3d ).

Unlike RbcL, the RbcS subunits are characterized by greater sequence diversity, with ≥30%similarity observed across species. Furthermore, plants and green algae contain families of differ-entially expressed RbcS isoforms, suggesting a regulatory function (75, 112). The common corestructure is a four-stranded antiparallel β sheet covered on one side by two helices (58) (Figure 3e).The small subunits are considered an evolutionary adaptation to the increasing oxygen levels inthe atmosphere, possibly functioning to stabilize the RbcL active sites and to improve the CO2/O2

specificity of form I Rubisco (3, 112). Although it does not participate directly in the formation ofthe active site, RbcS is required for catalytic activity through long-range effects (5, 17). The vari-ation among RbcS subunits of different species predominates in the loop between strands βA andβB and at the C terminus (Figure 3e). For example, the RbcS subunits of red-type Rubiscos featurean additional C-terminal β hairpin (strands βE and βF) that is not present in green-type Rubiscos.In the red-type Rubisco complex, the β hairpins of four RbcS subunits combine to form an eight-stranded β-barrel structure, lining the pore at both ends of the RbcL8 core complex (Figure 3b).The contacts between RbcS subunits in green-type Rubiscos are less extensive (Figure 3a).

Reactions Catalyzed by Rubisco

The Rubisco reaction sequence involves five steps for the final production of two molecules of3PGA: enolization (deprotonation) at C3, carboxylation at C2, hydration at C3, cleavage of theC2-C3 bond, and stereospecific protonation of one of the products (2) (Figure 4). Several ofthese steps involve acid-base chemistry. The first step of deprotonation at C3 of RuBP yields anunstable enediolate intermediate. Electrophilic addition of CO2 generates a branched six-carbonintermediate, which is hydrolytically cleaved into two molecules of 3PGA. The reaction thusresults in a net gain of organic carbon. However, the enediolate intermediate is also susceptibleto reaction with molecular oxygen, giving rise to a hydroperoxy derivative that breaks downto 2PG and 3PGA, thus precluding net carbon gain (Figure 4). Erroneous reprotonation ofthe enediolate intermediate gives rise to the misfire sugar phosphate D-xylulose-1,5-bisphosphate(XuBP) (Figure 4). D-Glycero-2,3-pentodiulose-1,5-bisphosphate (PDBP) and 2-carboxytetritol-1,4-bisphosphate (CTBP) are potential misfire products of the oxygenase reaction (91) (Figure 4).Binding of these sugar phosphates to Rubisco active sites impedes catalysis, diminishing the active

←−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−Figure 3Structures of form I Rubisco. (a) Structure of the green-type form IB Rubisco holoenzyme (from Nicotianatabacum; PDB 4RUB) (115). The RbcL8 core is shown in a surface representation, and the RbcS subunits areshown in teal in a ribbon representation. (b) Structure of the red-type form IC Rubisco holoenzyme (fromthe bacterium Alcaligenes eutrophus, also known as Ralstonia eutropha; PDB 1BXN) (42). The side view is across section to highlight the central barrel formed by the β-hairpin extensions (EF loop; see panel e) of theRbcS subunits that mediate assembly. (c) Ribbon representation of the antiparallel RbcL dimer unit of N.tabacum in complex with the transition-state analog CABP. The N- and C-terminal domains are colored inyellow and green, respectively. CABP is shown in a ball-and-stick representation. (d ) Substrate-bindingpocket with bound CABP, showing the boxed area from panel c. The catalytic Mg2+ ion is highlighted as apurple sphere. The loop 6 and C-terminal tail of RbcL, indicated in cyan and blue, respectively, are layeredon top of CABP. (e) Comparison of green- and red-type RbcS subunits, showing the structures from N.tabacum (PDB 4RUB) and A. eutrophus (PDB 1BXN). The divergent AB and EF loops are indicated.Abbreviations: CABP, 2-carboxyarabinitol-1,5-bisphosphate; PDB, Protein Data Bank; Rubisco,ribulose-1,5-bisphosphate carboxylase/oxygenase.


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RuBP Enediolate

2 × 3PGAC

C3

C

O

H

H

CH2OPO32–

CH2OPO32-

OH

OH

– H+

+ H+

C

C

C

–O

H

CH2OPO32–

CH2OPO32-

OH

OH

Enolization+ CO2

Carboxylation

Hydration Bondcleavage

Stereospecificprotonation

C

C

CH

CH2OPO32–

CH2OPO32–

O

OH

HO COO-

Carboxyketone

C

CH

CH2OPO32–

OH

COO–

XuBP

– H++ H+

C

C3

C

O

H

CH2OPO32–

CH2OPO32–

OH

OH H

+ O2

Oxygenation

Peroxyketone

C

C

CH

CH2OPO32–

CH2OPO32–

O

OH

HO O OH

+

1 × 3PGA1 × 2PG

C

CH2OPO32–

O–O

C

CH

CH2OPO32–

OH

COO–

Misprotonation

– H2O2

C

C

CH

CH2OPO32–

CH2OPO32–

OH

O

C

CH2OPO32–

CH

CH2OPO32–

OH

HOC

C

CH

CH2OPO32–

CH2OPO32–

O

OH

HO H

PDBPKABP CTBP

O?

COO-

Figure 4Reactions catalyzed by Rubisco. The critical Rubisco carboxylase and oxygenase reaction intermediates and products are shown. Theproductive carboxylation reaction comprises five steps (enolization, carboxylation, hydration, bond cleavage, and stereospecificprotonation) and results in the formation of two molecules of 3PGA, whereas the oxygenase reaction results in the formation of onemolecule of 3PGA and one molecule of 2PG, the latter of which is toxic to chloroplasts. The first intermediate common to bothreactions is the highly reactive enediolate obtained after abstraction of a proton from the third carbon (C3) of RuBP. Misprotonation ofthe enediolate results in the production of the Rubisco inhibitory sugar phosphate XuBP. The oxygenation reaction intermediateperoxyketone can also lead to the production of the inhibitory sugar phosphates KABP, PDBP, and CTBP. Inhibitory sugar phosphatesare indicated in red. Note that RuBP, the natural substrate, can also act as an inhibitor when binding to noncarbamylated Rubisco.Abbreviations: CTBP, 2-carboxytetritol-1,4-bisphosphate; KABP, 3-ketoarabinitol-1,5-bisphosphate; PDBP,D-glycero-2,3-pentodiulose-1,5-bisphosphate; 2PG, 2-phosphoglycolate; 3PGA, 3-phosphoglycerate; Rubisco,ribulose-1,5-bisphosphate carboxylase/oxygenase; RuBP, ribulose-1,5-bisphosphate; XuBP, D-xylulose-1,5-bisphosphate.

Chaperonin:a member of a class ofstructurally relatedATP-regulatedmolecular chaperonesthat form large,double-ringcomplexes, providing anano-compartment fora single nonnativeprotein to fold inisolation

pool of Rubisco (91). Inhibitory binding of RuBP to noncarbamylated Rubisco sites is also acommon impediment to catalysis (97, 110).

RUBISCO SUBUNIT FOLDING

The RbcL subunit is a classical example of the subset of proteins that require assistance by chap-eronins for folding. Chaperonins are ATP-regulated double-ring complexes that provide a nano-compartment for a single molecule of nonnative polypeptide to fold in isolation, unimpaired byaggregation. They are essential for protein folding in all domains of life and are found in thebacterial, archaeal, and eukaryotic cytosol, as well as in the chloroplast stroma and mitochon-drial matrix (46, 72) (Table 1). The chaperonins in bacteria (GroEL), chloroplasts (Cpn60), andmitochondria (Hsp60) are closely related and are classified as group I chaperonins, reflective ofthe endosymbiotic origin of chloroplasts and mitochondria. They cooperate with the lid-shapedcofactors GroES in bacteria, Cpn10/Cpn20 in chloroplasts, and Hsp10 in mitochondria (46)

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Table 1 Factors involved in biogenesis and metabolic maintenance of Rubisco

ProteinaMW(kDa) Oligomeric state Function Organisms References

Folding

Bsd2 (Znj2) ∼10 Unknown Binding of nascentRbcL on ribosomes

Red algae, green algae,plants

20, 24

GroEL ∼57 Tetradecamer RbcL folding Bacteria, cyanobacteria,red algae

38, 70

GroES ∼10 Heptamer RbcL folding Bacteria, cyanobacteria,red algae

38, 70

Cpn60α (RbcLbinding protein α)

∼60 Tetradecamer withCpn60β at 1:1 ratio

RbcL folding Green algae, plants 83, 130, 132

Cpn60β (RbcLbinding protein β)

∼60 Tetradecamer withCpn60α at 1:1 ratio

RbcL folding Green algae, plants 83, 130, 132

Cpn10 ∼10 Homo-heptamer ormixed assembly withCpn20

RbcL folding Green algae, plants 63

Cpn20 ∼20 Homo-tetramer ormixed assembly withCpn10

RbcL folding Green algae, plants 11, 45

Cpn23 ∼23 Homo-tetramer ormixed assembly withCpn10

RbcL folding Green algae 130

Assembly

RbcX ∼15 Dimer Assembly of RbcL2 andRbcL8 core

Cyanobacteria, greenalgae, plants

15, 60, 70, 107

Raf1 ∼40–46 Dimer Assembly of RbcL2 andRbcL8 core

Cyanobacteria, greenalgae, plants

34, 44, 59

Raf2 (acRAF) ∼9; ∼18inplants

Dimer Assembly of RbcL8S8 Autotrophic bacteria,cyanobacteria, greenalgae, plants

33, 137

Metabolic maintenance

Rca-IC (CbbX) ∼35 Hexamer AAA+ protein thatreactivates inhibitedRubisco

Autotrophic bacteria,red algae

78

Rca-IA (CbbQ) ∼30 Hexamer AAA+ protein thatreactivates inhibitedRubisco

Autotrophic bacteria 116, 129

Rca-IA adaptor(CbbO)

∼82–88 Monomer Adaptor protein forCbbQ

Autotrophic bacteria 129

Rca-IB ∼42 Hexamer AAA+ protein thatreactivates inhibitedRubisco

Green algae, plants 12, 43, 114

XuBPase (CbbY) ∼26 Monomer XuBP phosphatase Autotrophic bacteria,red algae, green algae,plants

16

(Continued )


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Table 1 (Continued )

ProteinaMW(kDa) Oligomeric state Function Organisms References

CA1Pase ∼48 Unknown CA1P and PDBPphosphatase

Plants 4

Abbreviations: AAA+, subgroup of the ATPases associated with various cellular activities; CA1P, 2′-carboxy-D-arabinitol-1-phosphate; MW, molecularweight; PDBP, D-glycero-2,3-pentodiulose-1,5-bisphosphate; Rubisco, ribulose-1,5-bisphosphate carboxylase/oxygenase; XuBP, D-xylulose-1,5-bisphosphate.aAlternative protein names are shown in parentheses.

(Table 1). The chloroplast chaperonin was initially named Rubisco large subunit binding pro-tein, based on findings that newly synthesized RbcL subunits interact with a large protein complexbefore forming the holoenzyme (9, 105). The chaperonins were initially proposed to mediate sub-unit assembly but were later shown to be required for protein folding (19, 88).

The Bacterial Chaperonin

The most extensively studied chaperonin system is the bacterial GroEL and its cofactor GroES(Table 1). GroEL is an∼800-kDa homo-oligomeric complex consisting of two heptameric ringsof ∼57-kDa subunits that are stacked back to back (Figure 5a). The subunits are composed ofthree domains formed by discontinuous sequence elements: an equatorial ATP-binding domain,an intermediate hinge domain, and an apical domain that forms the ring opening and containsthe binding surfaces for substrate protein and GroES (106). GroES is a single heptameric ringof ∼10-kDa subunits that binds to the ends of the GroEL cylinder and undergoes binding andrelease cycles regulated by the GroEL ATPase (46) (Figure 5b). Typical substrates of GroEL,such as RbcL, populate aggregation-prone folding intermediates that bind to GroEL via exposedhydrophobic amino acid residues. Functional reconstitution of Rubisco using GroEL-GroESin vitro was first achieved with the dimeric protein (form II) from Rhodospirillum rubrum (38).Studies over the last 25 years revealed the chaperonin mechanism (reviewed in 46) (Figure 5c).First, nonnative substrate protein is captured by the open ring of the GroEL-GroES complex.Subsequent binding of ATP and GroES, accompanied by dramatic conformational changes of theGroEL subunits, results in the release of bound substrate protein into an enclosed cage formed byGroEL and GroES. This cage is hydrophilic in nature and thus permissive for folding; it is largeenough for proteins up to ∼60 kDa. The substrate is allowed to fold inside the cage for the timeit takes the GroEL ring to hydrolyze its seven ATP molecules (a few seconds, depending on thetemperature). Following hydrolysis, ATP binding to the opposite ring causes the dissociation ofthe GroEL-GroES complex and release of the native protein.

Whereas the folded RbcL subunits of form II Rubisco rapidly assemble to functional dimersupon release from the chaperonin (19), the RbcL subunits of cyanobacterial form I Rubisco areunable to assemble spontaneously into the RbcL8 core complex. As discussed below, this failureto assemble is due to intrinsic properties of RbcL and is overcome only with the assistance ofspecific assembly factors. By contrast, RbcS refolds spontaneously in vitro to a state competent toassemble with the RbcL8 complex (6).

The Chloroplast Chaperonin

The plastid chaperonin system is typically encoded in the nucleus, and the proteins are importedfrom the cytosol. Chloroplast chaperonins differ from bacterial and mitochondrial group Ichaperonins in that they are hetero-oligomeric, consisting of two distinct isoforms of Cpn60

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a

137 Å

GroEL

c

146 Å

b GroES

77 Å

35 Å

7 ADP

7 ATP7 Pi 7 ADP

7 ATP

GroEL

GroES

~2–7 s

Collapsedintermediate

Native protein

ADP ADPATP ATP

d Cpn60β14 Cpn60α7β7e

Cpn23Cpn20Cpn10

Figure 5Chaperonin structure and function. (a,b) Surface representations of the GroEL tetradecamer complex (panel a; PDB 1J4Z) (18) and theGroES heptamer (panel b; PDB 1AON) (147). The GroEL subunits are shown in gray; the apical, intermediate, and equatorialdomains of one subunit of GroEL are shown in yellow, blue, and red, respectively. (c) The generic GroEL-GroES protein-foldingreaction cycle. The substrate protein is bound as a collapsed folding intermediate by the open GroEL ring of the asymmetricalGroEL-GroES-ADP complex. Binding of ATP to the substrate-bound ring causes a conformational change in the apical domains thatresults in the exposure of the GroES-binding residues. Binding of GroES causes substrate displacement into an enclosed folding cage.ADP and GroES dissociate from the opposite ring together with previously bound substrate (not shown). The newly encapsulatedsubstrate is free to fold in the GroEL cavity during the time needed to hydrolyze the bound ATP molecules (∼2–7 s, depending on thetemperature). ATP binding followed by GroES binding to the opposite ring triggers ADP release and GroES dissociation, resulting inthe release of the native protein. (d ) Possible subunit arrangements of the chloroplast chaperonin Cpn60. Chloroplast chaperonins arehetero-oligomeric, consisting of two distinct isoforms, Cpn60α and Cpn60β. The subunit composition of the heptameric rings in vivois not clear. The β isoforms form homo-oligomers in vitro, whereas the α subunits assemble only in the presence of β subunits,resulting in a 1:1 stoichiometric complex. Two possible configurations with this stoichiometry are shown. (e) Possible subunitarrangements of the chloroplast Cpn60 cochaperones. In addition to the GroES-like Cpn10, plastids contain Cpn20, which is a tandemrepeat of Cpn10 units. Chlamydomonas reinhardtii encodes two Cpn20 homologs (Cpn20 and Cpn23). Shown are various configurationsand/or combinations of Cpn10, Cpn20, and Cpn23, mimicking the sevenfold symmetry of bacterial GroES. In the case of a Cpn20tetramer, one of the eight Cpn10 units does not participate in the interaction with Cpn60α7β7 (130). Abbreviations: PDB, ProteinData Bank; Pi, inorganic phosphate. Panel c modified from Reference 56 with permission.

subunits, Cpn60α and Cpn60β (83) (Table 1). These isoforms share∼50% amino acid sequenceidentity and occur at a 1:1 stoichiometry in the tetradecameric complex (128, 132). The subunitarrangement in the heptameric rings in vivo is not clear (Figure 5d ). The β subunits can assembleinto homo-oligomeric chaperonin complexes upon recombinant expression in Escherichia coli,but the α subunits assemble only in the presence of β subunits, resulting in a 1:1 stoichiometricheptadecameric complex. The existence of α- and β-subunit paralogs contributes additionalcomplexity. For example, the green alga C. reinhardtii expresses one Cpn60α and two Cpn60β

subunits (Cpn60β1 and Cpn60β2) (108, 149), and Arabidopsis thaliana encodes two Cpn60α


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and four Cpn60β paralogs, which vary in relative abundance (132). Cpn60α2 single mutantsand Cpn60β2/Cpn60β3 double mutants in A. thaliana display reduced growth and an albinophenotype, consistent with a defect in Rubisco biogenesis (93, 117). Coexpression of Cpn60α

and Cpn60β from C. reinhardtii functionally replaces GroEL in E. coli (8), indicating that thechloroplast chaperonin system functions by the same mechanism as GroEL-GroES.

The complexity in subunit composition also extends to the plastid GroES homologs. In additionto the GroES-like Cpn10 (∼10 kDa) (63), plastids contain Cpn20 (∼20–24 kDa), which is a tandemrepeat of Cpn10 units (11) (Figure 5e, Table 1). Moreover, C. reinhardtii encodes two Cpn20homologs (Cpn20 and Cpn23) that differ mainly in the length of the linker between Cpn10 units(130). A. thaliana encodes only one Cpn20 but two Cpn10 isoforms (63). As reported recently,the Cpn10 and Cpn20 homologs of C. reinhardtii function only as hetero-oligomers in vitro.A combination of three Cpn10 and two Cpn23 proteins (Figure 5e), mimicking the sevenfoldsymmetry of GroES, proved to be most efficient in folding RbcL subunits of form II Rubisco incooperation with GroEL (41, 130). By contrast, both homo- and hetero-oligomeric complexes ofA. thaliana cochaperones were functional in Rubisco reconstitution with recombinant A. thalianaCpn60α7β7. In the case of a Cpn20 tetramer, one of the eight Cpn10 units apparently did notparticipate in the interaction with Cpn60α7β7 (130) (Figure 5e). The diversity in both Cpn60and the Cpn10/Cpn20 cofactors provides for combinatorial possibilities, presumably adjustingthe chaperones to protein subsets among the ∼3,000 chloroplast proteins (89).

The majority of plastid proteins, including RbcS, are synthesized in the cytosol and must beimported (50). Although RbcS is able to fold spontaneously in vitro, it may require chaperoneassistance for folding following its translocation across the outer and inner chloroplast envelopes.A strong candidate is the stromal Hsp70, which is known to assist the folding of proteins importedinto chloroplasts (128). Similarly, newly synthesized RbcL within the chloroplast may first interactwith Hsp70 and then be transferred to Cpn60, based on multiple reports that the Hsp70 andchaperonin systems act coordinately in protein folding (56, 128). Additional proteins may beinvolved in this step, including the stromal protein bundle sheath defective 2 (Bsd2) (20, 104). Bsd2is a cysteine-rich ∼10-kDa protein found in plants, green algae, and red algae and has sequencesimilarity to the zinc-finger domain of the Hsp70 cochaperone, DnaJ (20). DnaJ triggers theATPase activity of DnaK, and its zinc-finger domain was implicated in interactions with clientproteins (118). The Bsd2 ortholog in C. reinhardtii, Znj2, comigrates with actively translatingribosomes (24), consistent with the proposed role of Bsd2 in stabilizing the nascent chain of RbcL(20). In vitro, Znj2 has a chaperone-like function in preventing protein aggregation (24).

CHAPERONES FOR RUBISCO ASSEMBLY

Although the term molecular chaperone was coined to describe a role in assisting oligomericprotein assembly, the vast majority of studies have focused on the function of chaperones inmediating protein folding (27). More recently, however, an increasing number of chaperones withspecific functions in the oligomeric assembly of protein complexes and macromolecular machineshave been reported (22, 27). These factors operate when the assembly of folded subunits by randomcollision is inefficient—for example, when folded subunits on their own are structurally dynamicand tend to misassemble, or when all components of a complex are not simultaneously available.

The form I Rubisco has emerged as a paradigm of assisted assembly. The assembly of theRbcL8S8 holoenzyme is thought to involve the formation of an RbcL8 core followed by thedocking of RbcS subunits. Spontaneous assembly is inefficient: Various cyanobacterial form IRubisco complexes have been expressed in E. coli, but only with low yields of 0.1–10% (65), withoverexpression of GroEL-GroES improving RbcL8S8 yield (39). However, Rubisco could not be

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Raf: Rubiscoaccumulation factor

reconstituted in vitro with GroEL-GroES alone, suggesting the requirement of additional factors.So far, three assembly factors for green-type form IB Rubisco—RbcX, Rubisco accumulation factor1 (Raf1), and Raf2—have been structurally and functionally characterized, and several others havebeen implicated in the assembly process (15, 17, 33, 34, 44, 59–61, 70, 107, 122, 123, 137) (Figure 2,Table 1). RbcX and Raf1 interact with RbcL downstream of chaperonin-assisted folding, whereasRaf2 is thought to function in assembly by interacting with RbcS. These chaperones are ATPindependent, and their interaction with the Rubisco subunits is dynamic in nature. RbcX andRaf1 occur in plants, green algae, and cyanobacteria containing form IB Rubisco and generallycoexist. Raf2 has so far been identified in eukaryotes containing form IB Rubisco (plants and greenalgae) and in organisms harboring green-type form IA Rubisco (Figure 2). Notably, no homologsof these factors have been identified in organisms containing red-type form I Rubisco. Instead,a critical role in assembly has been reported for the red-type RbcS, which has a C-terminal β-hairpin extension not present in green-type RbcS (52) (Figure 3e). Whether the homologs ofCpn60 and DnaK uniquely encoded in the chloroplast genome of red algae also play a specializedrole in Rubisco assembly remains untested.

RbcX

Coexpression of RbcX enhances the biogenesis of various cyanobacterial Rubiscos in E. coli (28,66, 86, 107). RbcX functions by stabilizing the antiparallel RbcL dimer, the unit for RbcL8 coreassembly, and dissociates from RbcL8 upon binding of RbcS (17, 70, 107). The rbcX gene islocated in the intergenic space between the rbcL and rbcS genes in the Rubisco operon of severalcyanobacterial species (65) and is conserved in plants and green algae. RbcX has been reported to beessential in Synechococcus sp. strain PCC7002 (86) but not in Synechococcus elongatus strain PCC7942(28). In the latter strain, the rbcX gene is located outside the Rubisco operon but still enhancesRbcL8S8 biogenesis when coexpressed with Rubisco in E. coli (28). Thus, the dependence on RbcXapparently varies among cyanobacterial species, perhaps owing to redundancy of assembly factors.The RbcX proteins of plants and green algae are encoded in the nucleus and have chloroplast-targeting sequences. They are classified as RbcX-I and RbcX-II, of which RbcX-I is more closelyrelated to cyanobacterial RbcX (15, 60, 107). Although C. reinhardtii and A. thaliana RbcX canfunctionally substitute for cyanobacterial RbcX (15, 61), a requirement of RbcX in plant Rubiscobiogenesis remains to be established.

Crystal structures of RbcX from several cyanobacterial species showed that RbcX is a homodi-mer of mostly α-helical ∼15-kDa subunits with a central cleft (107, 122, 123) (Figure 6a). Eachsubunit consists of four α helices that form a four-helix bundle at one end. The two long α4helices from each protomer align antiparallel, with a 60◦ kink at their midpoint, resulting in thehelix bundles being located at opposite ends of the arc-shaped dimer. The residues in the centralcleft are highly conserved and hydrophobic in character (Figure 6b). Similar structures have beenreported for RbcX homologs of C. reinhardtii and A. thaliana (44, 60).

Biochemical and structural analysis has shown that the sequence motif EIKFE(F/Y)X, foundat the C terminus of many RbcL sequences (Figure 2), binds within the central cleft of RbcX.The Phe/Tyr side chains of RbcL protrude into hydrophobic pockets in the RbcX cleft (107).Polar residues at the peripheral regions of the RbcX dimer are also required for the assemblyprocess (107) (Figure 6b). The crystal structure of an end-stage assembly intermediate showedthat RbcX functions as a molecular clamp for the RbcL2 units (17). In this structure, four RbcXdimers are bound at the top and four at the bottom of the RbcL8 core (Figure 6c). Each RbcXbinds the C-terminal motif of one RbcL subunit, and one of its peripheral regions contacts theRbcL subunit adjacent in the dimer.


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The use of RbcX in conjunction with GroEL-GroES allowed the first in vitro reconstitutionof cyanobacterial form I Rubisco from S. elongatus strain PCC6301 and dissection of the assemblymechanism (70). These experiments indicated that RbcX prevents rebinding of the folded RbcLmonomer to GroEL by stabilizing RbcL2 units and shifting the equilibrium to RbcL8RbcX8

complexes (Figure 6d ). Binding of RbcS triggers conformational changes in RbcL and facilitatesthe displacement of RbcX, presumably via transient intermediates with both RbcX and RbcS boundto RbcL8. RbcX function critically depends on the dynamic nature of its interactions with RbcL(central cleft and peripheral region) (107). Indeed, a heterologous RbcX with a higher affinity forS. elongatus strain PCC6301 RbcL could not be displaced by RbcS (70).

a

Q51

Q51I50

I50Y17 Y20

Y20Y17E32

Q29

E32Q29

E107R108

E108R107

b

N

C

α1

α2

α3

α4

90°α1

α2

α3

α4

c

90°

100 Å

175 Å

Side view Top view

GroEL

RbcL

RbcX RbcS

RbcLRbcX

d

RbcX

RbcL2RbcX2 RbcL8RbcX8 RbcL8RbcS8

Similarity score

0% 50% 100%

65 Å

35 Å

GroES

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Raf1

The chloroplast Raf1 was identified from the Photosynthetic Mutant Library, a collection of∼2,000 maize mutants that display chlorophyll defects (113). Maize lines lacking Raf1 areRubisco deficient and seedling lethal, although Rubisco is transcribed and translated. Cross-linking experiments showed an association of Raf1 with RbcL, suggesting a role in assembly(34). Raf1 homologs appear to be present in all eukaryotic and cyanobacterial organisms that alsohave RbcX. Coexpression of cyanobacterial Raf1 with Rubisco in E. coli promoted Rubisco assem-bly (59). Interestingly, the formation of functional hybrid Rubisco consisting of A. thaliana RbcLand tobacco RbcS in tobacco plants was improved by coexpression of A. thaliana Raf1, consistentwith coevolutionary adaptation of Raf1 with its cognate RbcL (139).

Recent structural and biochemical analysis revealed that Raf1, like RbcX, is a functional dimer(44, 59, 139). Each ∼40–46-kDa protomer consists of an N-terminal α-helical domain and a C-terminal β-sheet dimerization domain separated by a flexible linker (44) (Figure 7a). Only oneface of each domain displays surface residue conservation among Raf1 homologs, and chemicalcross-linking coupled to mass spectrometry identified these surfaces as RbcL interaction sites,with the α-helical domains making the major contribution to binding (44) (Figure 7b). A com-plex consisting of RbcL8 and four Raf1 dimers was identified as the end-state intermediate ofRaf1-mediated Rubisco assembly. Analysis of this complex by electron microscopy and single-particle reconstruction showed that each Raf1 dimer embraces one RbcL2 unit (44) (Figure 7c).The β domains are arranged around the equator of the RbcL8 complex, whereas the α domainscontact the top and bottom edges of the RbcL dimer. In vitro reconstitution of cyanobacte-rial Rubisco with GroEL-GroES and Raf1 allowed almost quantitative formation of the activeholoenzyme. In the presence of Raf1, the folded RbcL is efficiently released from GroEL andforms various assembly intermediates with Raf1, including an RbcL2 unit complexed with oneRaf1 dimer (Figure 7d ). Assembly is completed by the displacement of Raf1 from RbcL8 byRbcS.

Interestingly, the RbcX and Raf1 assembly factors perform the same function in stabiliz-ing the RbcL2 unit but do so by using different interaction sites on RbcL (Figures 6d and7d ). An important question therefore concerns whether RbcX and Raf1 act in parallel as-sembly pathways or cooperate in vivo to achieve efficient assembly at a biologically relevanttimescale.

←−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−Figure 6The RbcX assembly chaperone. (a) Ribbon representation of the cyanobacterial RbcX dimer (PDB 2PEN) (107). Two perpendicularviews are shown. The two protomers are indicated in orange and gold. (b) Surface conservation of cyanobacterial RbcX. The similarityscores from an alignment of 151 sequences of cyanobacterial RbcX in the Pfam database were plotted onto the accessible surface of theRbcX dimer. Sequence conservation is displayed as a color gradient, with highly conserved residues shown in magenta and variableregions shown in cyan. The positions of conserved surface residues are also indicated. (c) Crystal structure of a trapped RbcL8RbcX8assembly intermediate (PDB 3RG6) (17). The RbcL8 core structure is shown in a surface representation, and the bound RbcX is shownin a ribbon representation. The C-terminal tails of the RbcL subunits are bound within the central hydrophobic cleft of RbcX, and theconserved polar residues at the RbcX corners are in contact with the N domain of the adjacent RbcL subunit in the antiparallel dimer.(d ) Model of RbcX-assisted assembly of green-type cyanobacterial form I Rubisco. Upon folding by the GroEL-GroES chaperonincomplex (with GroEL in gray and GroES in light blue), the released RbcL subunit exposes the flexible C-terminal RbcL peptide.Whether RbcX (orange) recognizes and binds the monomeric or the spontaneously dimerized RbcL subunits (beige) is not clear. Theantiparallel RbcL dimer is stabilized by RbcX dimers acting as molecular staples. The RbcL2RbcX2 units subsequently assemble intothe RbcL8RbcX8 complex, in which a large portion of the RbcS (teal ) binding interface is preformed. RbcS binding structures theRbcL N terminus and the 60s loop of RbcL, causing displacement of RbcX and formation of the functional Rubisco holoenzyme.Abbreviations: E, glutamic acid; I, isoleucine; PDB, Protein Data Bank; Q, glutamine; R, arginine; Rubisco, ribulose-1,5-bisphosphatecarboxylase/oxygenase; Y, tyrosine. Panel b modified from Reference 107 with permission from Elsevier.


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Raf2

Raf2 homologs have been identified in plants (33) and in a subset of cyanobacteria expressingform IA Rubisco, where the raf2 gene is part of the α-carboxysome operon together with rbcLand rbcS (137). Mutation of Raf2 in maize causes seedling lethality and Rubisco deficiency (33),a phenotype similar to the mutation of Raf1, albeit less severe (34). Raf2 proteins share sequence

a b

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44 Bracher et al.

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homology to pterin-4α-carbinolamine dehydratase (PCD) enzymes but are catalytically inactive(81, 137). The crystal structure of Raf2 from Thiomonas intermedia shows a dimer of ∼9.5-kDasubunits with the characteristic PCD fold but the active-site cleft being disrupted (137). Thedimer exhibits a concave surface that is conserved (Figure 7e). Eukaryotic Raf2 homologs areconsiderably larger (∼18.5 kDa), carrying a conserved N-terminal extension (33). In plants, theprotein interacts with RbcS and to a lesser extent with RbcL (33), suggesting a possible role inchaperoning RbcS upon import and assembly with RbcL. Co-overexpression of cyanobacterialRaf2 and GroEL-GroES with Rubisco subunits in E. coli improved the yield of assembled Rubiscocompared with overexpression of GroEL-GroES alone (137). The exact mechanism by whichRaf2 promotes Rubisco biogenesis remains to be clarified.

Red-Type RbcS

The absence of the assembly factors described above from the genomes of organisms containingred-type form I Rubisco (Figure 2) suggests an alternative mechanism of assembly. Indeed, thered-type Rubisco of the proteobacterium Rhodobacter sphaeroides folds and assembles upon recom-binant expression in E. coli with high yield (52). Moreover, the GroEL-GroES chaperonin systemis sufficient to reconstitute R. sphaeroides Rubisco in vitro. However, the folded RbcL subunits as-semble only in the presence of RbcS (52), with the C-terminal β-hairpin extension of the red-typeRbcS playing a critical role (52). The β hairpins mediate RbcS-RbcS contacts, forming β-barrelstructures at the top and bottom of the holoenzyme solvent channel (Figure 3b). The detailedmechanism of RbcS-mediated assembly of RbcL remains to be investigated. The red-type RbcSwas also able to assemble green-type cyanobacterial RbcL in vitro, replacing the requirementfor RbcX or Raf1. However, the resulting heterologous complex was enzymatically inactive (52).Why the green-type enzymes have evolved a dependence on auxiliary assembly factors, ratherthan using the simpler RbcS-mediated mechanism, is an interesting question. An important pointis that in eukaryotic red-type organisms (nongreen algae), both rbcL and rbcS are plastid encoded,along with dnaK and cpn60 gene copies in red algae, whose products are presumably involved in

←−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−Figure 7The Rubisco accumulation factor chaperones. (a) Crystal structures of the C-terminal dimerization domain(β domain) (PDB 4WT3) and N-terminal α domain (PDB 4WT4) of Raf1 from Arabidopsis thaliana (44).The protomers in the β-domain dimer are shown in blue and cyan. (b) Surface conservation in the Raf1βdimer and Raf1α. The similarity scores from an alignment of 151 sequences of Raf1β and Raf1α in thePfam database were plotted onto the accessible surface of the Raf1β dimer and Raf1α. Sequenceconservation is displayed as a color gradient, with highly conserved residues shown in magenta and variableregions shown in cyan. (c) Three-dimensional reconstruction of the RbcL8Raf14 complex fromnegative-stain electron microscopy images (EMDB EMD-3053). Side and top views of the structural modelare shown. Rigid-body domain fitting of the RbcL8 core and one Raf1 in the complex is superposed. TheRaf1 dimer subunits (ribbon representation) are shown in blue and cyan, and the RbcL8 core (schematicrepresentation) is shown in pale yellow. (d ) Model of Raf1-mediated Rubisco assembly of green-typecyanobacterial form I Rubisco. After folding by the GroEL-GroES chaperonin system (with GroEL in grayand GroES in light blue), the released RbcL subunits (beige) either dimerize spontaneously or do so withassistance from Raf1 (dark blue). The antiparallel RbcL dimer is stabilized by Raf1 and is in dynamicequilibrium with higher oligomers up to RbcL8Raf14 complexes. RbcS (teal ) binding shifts the equilibriumtoward holoenzyme formation. (e) Crystal structure of the bacterial Rubisco accumulation factor Raf2 (PDB4LOW) (137). The protomers in the dimer are shown in pink and red. On the right, surface conservation inbacterial Raf2 homologs is indicated, as in panel b. Abbreviations: EMDB, Electron Microscopy Data Bank;PDB, Protein Data Bank; Raf, Rubisco accumulation factor; Rubisco, ribulose-1,5-bisphosphatecarboxylase/oxygenase. Panel b modified from Reference 44.


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Rubisco biogenesis (1, 138). Understanding the biogenesis requirements of these Rubiscos is ofbiotechnological interest because of their superior CO2/O2 specificity and potential for improvingplant productivity (71, 90, 140).

The Challenge of Plant Rubisco Reconstitution

Despite the recent success in reconstituting cyanobacterial form I Rubisco with chaperonins andeither RbcX or Raf1, the Rubisco from plants has resisted efforts to generate the assembledholoenzyme in vitro or upon recombinant expression in E. coli with these factors. This is surprisingin light of the high sequence and structural similarity of the cyanobacterial and plant enzymes.It is possible that the full spectrum of auxiliary factors required by the plant enzyme has notbeen identified (144). The high degree of macromolecular crowding in the chloroplast stroma,a condition that is difficult to mimic in vitro, may also play an important role in facilitating theassembly process in vivo (150).

METABOLIC MAINTENANCE

Once assembled and activated by carbamylation, Rubisco continues to depend on accessory factorsto maintain functionality. Partial or complete deactivation may be caused by the spontaneous loss ofMg2+ and the carbamyl prosthetic group in the active site. Production of uncarbamylated enzymescan be facilitated in vitro by elevated temperature and extraction in the absence of RuBP, CO2, andMg2+ ions (4, 77, 91). Decarbamylation allows autoinhibition by binding of the substrate RuBP(Figure 8a). This inhibition can be sustained in vivo and in vitro under conditions where RuBP

−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−→Figure 8Rubisco inactivation and reactivation. (a) Regulation of Rubisco activity and inhibition by sugar phosphates,showing the noncarbamylated enzyme (E), the carbamylated and Mg2+ ion–bound enzyme (ECM), the sugarphosphate–inhibited E form (EI), and the sugar phosphate–inhibited ECM form (ECMI). (b) Structure ofthe prokaryotic form IC Rca from Rhodobacter sphaeroides. The crystal structure of the monomer (PDB 3SYL)is shown in a ribbon representation on the left. The α/β and α-helical subdomains are shown in blue andteal, respectively, and an N-terminal extension is shown in purple. The pore loop is also indicated. Boundsulfates from the precipitant are shown in ball-and-stick form and represent the nucleotide binding site at theinterface of the two domains and the RuBP binding site in the α-helical subdomain. The middle and rightsubpanels show top and side views, respectively, of an electron microscopy reconstruction of the Rca hexamer,with alternating subunits shown in two shades of red (EMDB EMD-1932, PDB 3ZUH). The positionsof bound ADP (cyan) and RuBP (orange) are also indicated. (c) Structure of the prokaryotic form IA Rca fromHalothiobacillus neapolitanus. The crystal structure of the monomer in the hexameric complex (PDB 5C3C)with bound ADP is shown in a ribbon representation on the left (116). The middle and right subpanels showtop and side views, respectively, of the hexamer superposed on an electron microscopy reconstruction of theRca isoform from Acidithiobacillus ferrooxidans (EMDB EMD-6477) (129). The alternating subunits are shownin two shades of violet, and the bound ADP is shown in cyan. (d ) Structure of eukaryotic form IB Rca fromNicotiana tabacum. The crystal structure of the monomer (PDB 3T15) is shown in a ribbon representationon the left. The middle and right subpanels show top and side views, respectively, of an electron microscopyreconstruction of the hexamer complex, with alternating subunits shown in two shades of green (EMDBEMD-1940, PDB 3ZW6). The unfilled electron microscopy density at the top of the hexamer probablyrepresents the N domain, which is not present in the crystal structure. The specificity helix in the α-helicalsubdomain that confers the ability of form IB Rca to discriminate between solanaceous and nonsolanaceousRubisco is also indicated in dark pink. Abbreviations: CA1P, 2′-carboxy-D-arabinitol-1-phosphate;EMDB, Electron Microscopy Data Bank; PDB, Protein Data Bank; PDBP, D-glycero-2,3-pentodiulose-1,5-bisphosphate; Rca, Rubisco activase; Rubisco, ribulose-1,5-bisphosphate carboxylase/oxygenase;RuBP, ribulose-1,5-bisphosphate; XuBP, D-xylulose-1,5-bisphosphate. Panel a modifiedfrom References 45 and 91; panel b modified from Reference 78; panel d modified from Reference 114.

46 Bracher et al.

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levels remain saturating (51, 110). Another cause of deactivation is the production of misfire sugarphosphates, such as XuBP (Figure 4), whose accumulation during assays in vitro leads to a gradualdecline in activity termed fallover. The higher rates of fallover measured in early studies in vitrowere shown to be not physiologically relevant but rather a consequence of PDBP impurities inRuBP preparations (53). Under low illumination, plants can also produce the nighttime inhibitor

Specificityhelix

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CO2 + Mg2+

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RuBP + CO2

CO2fixation

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Eukaryotic Rca (form IB)

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CA1P: 2′-carboxy-D-arabinitol-1-phosphate

Rca: Rubisco activase

AAA+: subgroup ofthe ATPasesassociated with variouscellular activities

2′-carboxy-D-arabinitol-1-phosphate (CA1P) to shut down Rubisco activity (4, 91). In all of theseinhibited states, Rubisco must be conformationally remodeled by a Rubisco activase (Rca) to regaincatalytic activity (96, 97) (Table 1).

Rca has been known since the 1980s (98) and was initially assumed to be restricted to plants.However, recent research has revealed that functionally equivalent but structurally unrelated Rcaproteins have evolved independently and are associated with red and green lineages of Rubisco(45, 77). Deletion of the Rca proteins in A. thaliana and R. sphaeroides results in severe photoau-totrophic growth defects (77). Rca proteins belong to a subgroup of the ATPases associated withvarious cellular activities (AAA) called AAA+. All Rca proteins share the AAA+ domain architec-ture, consisting of an N-terminal α/β-nucleotide-binding subdomain and a C-terminal α-helicalsubdomain, and form six-membered ring complexes (Figure 8b–d ). Members of this family ofproteins frequently act by threading extended sequences or loop segments of their target proteinsinto the central pore of the hexamer (111). In addition to Rca, photosynthetic organisms expressspecific phosphatases for the removal of inhibitory sugar phosphates (4, 16). The machineriesfor Rubisco maintenance are prime examples of metabolite damage repair, ensuring the timelyelimination of inhibitory and toxic side products (69).

Prokaryotic Rca

Prokaryotic Rca was only recently discovered in the proteobacterium R. sphaeroides, which containsred-type Rubisco (78); in this species, it is encoded by the cbbX gene in the Rubisco operon (37). Thestructural and functional analysis of the R. sphaeroides Rca (RsRca) provided critical insights intothe mechanism of Rubisco remodeling. The RsRca subunit (∼35 kDa) is composed of an AAA+core module with a compact α-helical extension at the N terminus (78) (Figure 8b). The activehexameric complex forms only in the presence of ATP and RuBP, the substrate of its target enzyme,Rubisco. In the absence of RuBP, RsRca forms spiral-shaped, high-molecular-weight assembliesthat are largely ATPase inactive and may represent a storage form (78). Thus, the generationof RuBP during photosynthesis would induce the conversion of RsRca into active functionalhexamers. The RuBP binding site is located in the α-helical subdomain at the bottom of thehexamer (Figure 8b). The hexamer exhibits a 25-A-wide central channel lined by canonical pore-loop residues (Tyr/Ile/Gly) (78). Biochemical and mutational analysis has shown that remodelingof Rubisco depends on the pore loops and the conserved top surface of the hexamer. Moreover,reactivation of Rubisco required the intact C-terminal sequence of RbcL, which is extended in red-type RbcL by∼5–10 residues relative to green-type RbcL. Binding to inhibited Rubisco stimulatesthe ATPase of RsRca approximately fourfold (78), in a manner dependent on both the RbcL Cterminus and the top surface of the RsRca hexamer. These findings suggest that RsRca docks ontoRubisco with its top surface and transiently pulls the C-terminal tail of RbcL into the central poreto facilitate opening of the active-site pocket and release the inhibitory sugar phosphate.

Another prokaryotic Rca was recently discovered in the chemoautotrophic bacteriaAcidithiobacillus ferrooxidans (129) and Halothiobacillus neapolitanus (116). The Rca proteins of thesespecies (AfRca and HnRca, respectively) function as bipartite complexes consisting of the hex-americ AAA+ protein CbbQ (∼30 kDa) (Figure 8c) and the Rubisco adaptor protein CbbO(∼82–88 kDa). CbbQ belongs to the MoxR group of prokaryotic AAA+ proteins, which are oftenassociated with von Willebrand factor A (VWA) domain proteins (145). CbbQ has a minimalAAA+ structure without additional extensions; the C-terminal subdomain consists of a five-helixbundle. Electron microscopy and crystal structures show that CbbQ forms compact hexamericrings with a diameter of 110 A (116, 129). In the electron microscopy structure of the CbbQ-CbbOcomplex, one CbbO subunit is bound to the CbbQ hexamer (129). Binding of CbbO stimulates

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the ATPase activity of CbbQ and is required for Rca activity. CbbO bears a flexibly tetheredC-terminal VWA domain with a metal ion–dependent adhesion site for Rubisco recognition.Notably, Tsai et al. (129) found that A. ferrooxidans contains two CbbQ-CbbO pairs, AfRcaI andAfRcaII, which are specific for the form I and form II (trimer of RbcL2 units) Rubiscos, respec-tively, in this organism. As with RsRca, interaction with the RbcL C terminus is necessary forATPase stimulation and Rca function, but so far no evidence for involvement of the CbbQ centralpore has been obtained. To our knowledge, this study (129) also provided the first evidence forthe requirement of an Rca by a form II Rubisco.

Heterocystous cyanobacteria (Anabaena and Nostoc strains) have been reported to contain pro-teins that combine an AAA+ module with an RbcS-like domain at the C terminus (67). TheirAAA+ module is highly related to plant Rca, but whether and (if so) how these proteins functionas bona fide Rca proteins remain to be investigated.

Eukaryotic Rca

Two forms of Rca coexist in the majority of green algae and plants, differing at their C terminus,with the α isoform having a slightly longer C-terminal tail than the β isoform (97). Dependingon the organism, the two isoforms are either encoded by separate genes or result from alternatesplicing. Eukaryotic Rca exists in a dynamic equilibrium of oligomeric states in vitro, but themain active form is the ring-shaped hexamer (12, 43, 47, 54, 64, 114). In contrast to prokaryoticRca, the eukaryotic Rca (∼48–50 kDa) contains an ∼5-kDa chloroplast-targeting sequence andan adjoining small N-terminal domain in addition to the AAA+ core module (Figure 8d ). Asin other AAA+ proteins, the N-terminal domain mediates target protein binding (29, 131). Itcooperates with a short helix (H9) in the α-helical subdomain, the so-called specificity helix (114).In tobacco, H9 interacts with residues Asp89 and Lys94 of the RbcL subunit (spinach numbering),located in the equatorial region of RbcL8S8, and confers the ability of Rca to discriminate betweensolanaceous and nonsolanaceous Rubisco (97, 134). Interestingly, the CbbO adapter protein of theprokaryotic AfRca recognizes the corresponding region (Asp82) on its cognate RbcL, a remarkableexample of convergent evolution (129).

Unlike prokaryotic Rca, the eukaryotic Rca is constitutively ATPase active and is regulatedby the intracellular ATP/ADP ratio or by reversible disulfide-bond formation in the C-terminalextension of the large isoforms in some plants (21, 97, 125). The crystal structures of Rca fromtobacco and A. thaliana exhibit helical subunit arrangements in the crystal lattice with six sub-units per turn (43, 114). The N domain and the unstructured C terminus are not resolved inthe structures. However, electron microscopy analysis of the tobacco Rca complex in solutiondemonstrated the hexameric ring assembly (114) and revealed the position of the N domains asextra densities at the top of the hexamer (Figure 8d ), forming the likely interface with the Rubiscocomplex. The mechanism of Rubisco remodeling by eukaryotic Rca is not understood. The centralpore of tobacco Rca (∼36 A) is wider than that of RsRca (∼25 A) and contains noncanonical poreloops (78, 114), yet mutational analysis implicates the pore loops in Rubisco remodeling (114).Moreover, the green-type RbcL lacks the extended C terminus that is required for the remodelingby red-type Rca. Thus, the sequence elements of RbcL that are threaded into the central pore ofRca, if any, remain to be identified.

Inhibitory Sugar Phosphatases

Remodeling of inhibited Rubisco by Rca catalyzes the release of inhibitory sugar phosphate fromthe active-site pocket. The released sugar phosphates retain high affinity for Rubisco and can


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effectively compete with RuBP (92). For example, XuBP is formed at a much higher rate thanother misfire products (Figure 4) and has an inhibitory constant (Ki) of ∼4.8 μM and ∼1.3 μMfor tobacco Rubisco and R. sphaeroides Rubisco, respectively (16, 92). Thus, XuBP and otherinhibitory sugar phosphates must be hydrolyzed by specific phosphatases to prevent their detri-mental buildup (91) (Table 1). The phosphatase that hydrolyses the phosphate at the C1 positionof XuBP (XuBPase) was only recently identified as the product of the cbbY gene (16) in the Rubiscooperon of R. sphaeroides, downstream of the gene for RsRca (cbbX) (37). Homologs of the CbbYprotein are widely distributed among photosynthetic organisms, including plants (133). Structuraland enzymatic analysis of XuBPase from R. sphaeroides and A. thaliana revealed how the enzymedistinguishes between XuBP and RuBP, which differ only in the stereochemistry at the C3 position(16).

XuBPase belongs to the haloacid dehalogenase hydrolase superfamily of enzymes (62). Likeother members of this superfamily, XuBPase (∼26 kDa) is a two-domain protein, consisting of acore domain harboring the catalytic residues and a flexibly attached cap domain, with the active-sitepocket located at the interface (Figure 9a). The structure of an inactive mutant showed that XuBPbinds in a distinct bent conformation, positioning the 1-phosphate group for efficient hydrolysis(16) (Figure 9b). Modeling RuBP in the same bent conformation would result in a steric clash,explaining the ∼100-fold-lower Michaelis constant (Km) for XuBP (∼30 μM) compared withRuBP (∼3 mM). Moreover, the turnover rate for XuBP is∼50-fold higher, resulting in a catalyticefficiency that is approximately four orders of magnitude greater (16). This allows XuBPase tohydrolyze micromolar concentrations of XuBP in the presence of millimolar RuBP, concentrationsof physiological relevance in chloroplasts (100). The resulting D-xylulose-5-phosphate (Xu5P)enters the Calvin-Benson-Bassham cycle and is converted back to RuBP (16) (Figure 9c).

The other well-characterized inhibitory sugar phosphate, CA1P, is hydrolyzed by a specificphosphatase in a light-dependent manner. CA1P has been found only in chloroplasts and is formedby phosphorylation of 2-carboxy-D-arabinitol during shading and darkness (91). CA1P bindstightly to the active site of carbamylated Rubisco. It is removed from the active site by Rca whenphotosynthesis resumes. At the same time, the CA1Pase is redox regulated and is activated asNADPH levels increase (91). CA1Pase also hydrolyzes PDBP, the relatively labile misfire productof the oxygenase reaction (4, 53).

PERSPECTIVES ON RUBISCO ENGINEERING

The growing world population and the use of agricultural land for the production of biofuelwill demand an ∼30–50% increase in agricultural productivity before 2050 (71, 87). The mainstrategy to increase crop yield has been to target the diverse aspects of photosynthesis, with thegoal of driving a new green revolution (95). Many of these targets are focused on overcomingthe limitations of Rubisco via a range of bioengineering objectives to enhance chloroplast CO2

levels (74, 94, 101) or to improve the catalytic properties of Rubisco itself (31, 90, 103). Recentadvances in understanding the machineries of Rubisco biogenesis and maintenance, as well as thedevelopment of new engineering procedures, suggest that the challenge of engineering a betterRubisco is not insurmountable.

Evolutionary Considerations

The major energetic investment photosynthetic organisms make in producing large quantities ofRubisco and/or concentrating CO2 around the enzyme imply that improving its catalytic efficiencyhas been an evolutionary challenge. Importantly, Rubisco’s structural framework and catalytic

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w.a

nnua

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a

XuBP

Capdomain

Coredomain

Mg

NC

b XuBP

O

P

O

O

OPO–

O–

O

OH

Mg

Asp8

Gly50

Gly51Glu17

Arg54

Asn10

His75

Lys78

Thr116

Thr115

Asn121

Ser118

Thr117 1

2

3

5

4

O–O–

c

RuBP

XuBPXu5P

Ru5P

CO2

2 × 3PGA

Pi

Rubisco

CbbY (+Rca)

ATP ADP

Ru5Pepimerase

Ru5P kinase

CBB cycle

60 Å

40 Å

HO

Figure 9Structure and function of XuBPase. (a) Crystal structure of a catalytically inactive XuBPase mutant (D10N)from the proteobacterium Rhodobacter sphaeroides (PDB 4UAT) (16) with XuBP trapped in the active-sitepocket, located at the interface of the cap ( yellow) and core (blue) domains. Bound XuBP is shown in green,and the catalytic Mg2+ ion is indicated by a sphere. (b) Hydrogen-bond interactions of XuBP with residuesof the cap ( yellow) and core (blue) domains. The mutant residue Asn10 is shown in red. In the wild-typeenzyme, residue Asp10 is positioned for efficient hydrolysis of the first phosphate of XuBP. (c) Functionalcooperation of XuBPase and Rca in maintaining Rubisco activity in the presence of the inhibitory misfireproduct XuBP. As soon as XuBP is released from the active-site pocket of Rubisco by Rca, CbbY hydrolyzesit to Xu5P. Xu5P is then converted to RuBP by the action of Ru5P epimerase and Ru5P kinase in the CBBcycle. Abbreviations: CBB, Calvin-Benson-Bassham; PDB, Protein Data Bank; 3PGA, 3-phosphoglycerate;Pi, inorganic phosphate; Rca, Rubisco activase; Rubisco, ribulose-1,5-bisphosphate carboxylase/oxygenase;RuBP, ribulose-1,5-bisphosphate; Ru5P, ribulose-5-phosphate; XuBP, D-xylulose-1,5-bisphosphate;XuBPase, XuBP phosphatase; Xu5P, D-xylulose-5-phosphate. Figure modified from Reference 16.

mechanism initially evolved in an oxygen-free atmosphere. The evolution of form I Rubisco inoxygen-tolerant organisms allowed the extraction of carbon from an atmosphere rich in oxygenbut also resulted in photorespiration and its associated energy and carbon costs, which may haveplaced further limitations on the Rubisco catalytic turnover rate (82, 124). The limited variation inRubisco sequences among phylogenetically distant organisms suggests that Rubisco has reachedan evolutionarily trapped state where further exploration of sequence space necessary to enhancecatalytic function may be restricted by a requirement for multiple complementary mutations (99,140).


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Rubi

sco

(Rbc

L 8S 8

)

RbcS

Activ

ated

Rubi

sco

RbcL

2Rb

cL8

CO2 +

Mg2+

cpn6

0α

cpn2

0

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er g

enes

cpn6

0β

cpn1

0

mRN

ARi

boso

me

Cyto

solic

nas

cent

cha

in–

bind

ing

chap

eron

esH

sp70

bsd2

rbcX

raf1

raf2

rbcS

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lear

geno

me

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40

Sign

alpe

ptid

e

rca

cbby

Chlo

ropl

ast s

trom

a

RuBP

CO2

O2

2 ×

3PG

A3P

GA

2PG

2PG

ase

Gly

cola

te

Mitochondrion

Peroxisome

Thyl

akoi

ds

Mis

prot

onat

ion

prod

uct:

XuBP

PDBP

CA1P

RuBP

Activ

eRu

bisc

o

Inac

tive

Rubi

sco

Rca

Phos

phat

ase

(e.g

., Xu

BPas

eor

CA

1Pas

e)

Fold

ed p

rote

in(e

.g.,

RbcS

)

Chap

eron

e-as

sist

ed

SPP

Raf2

?

Tic/

Toc

com

plex

Inhi

bito

rysu

gar p

hosp

hate

Non

inhi

bito

rypr

oduc

t

hsp7

0hs

p40

Cyto

sol

Oth

er c

ytos

olic

cha

pero

nes

(e.g

., H

sp90

or 1

4-3-

3 pr

otei

ns)

RbcX

?Ra

f1

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ropl

ast

geno

me

(10–

100

copi

es)

rbcL

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0αβ

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0/20

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?

mRN

A

Ribo

som

e

Nas

cent

cha

in–

bind

ing

chap

eron

es

rbcL

Hsp

70

Hsp

40

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E

RbcS

Fold

ing

chap

eron

es

Ass

embl

y ch

aper

ones

Met

abol

ic re

pair

fact

ors

P i

Figu

re10

Mul

tiplic

ityof

fact

ors

invo

lved

inth

ebi

ogen

esis

and

mai

nten

ance

ofeu

kary

otic

gree

n-ty

peR

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ubis

cobi

ogen

esis

requ

ires

coor

dina

tion

ofcy

toso

lican

dch

loro

plas

ttra

nsla

tion.

The

Rbc

Lsu

buni

tis

enco

ded

inth

ech

loro

plas

tand

synt

hesi

zed

onri

boso

mes

inth

ech

loro

plas

tstr

oma.

Cha

pero

nes

invo

lved

inth

efo

ldin

gof

new

lysy

nthe

size

dR

bcL

are

show

nin

yello

w,a

ndfa

ctor

sm

edia

ting

the

asse

mbl

yof

Rbc

L8

core

com

plex

esar

esh

own

inor

ange

.Mos

tchl

orop

last

prot

eins

,inc

ludi

ngth

efo

ldin

gan

das

sem

bly

fact

ors,

asw

ella

sR

bcS

(gre

en),

are

enco

ded

inth

enu

cleu

san

dsy

nthe

size

don

cyto

solic

ribo

som

esw

itha

chlo

ropl

ast-

targ

etin

gse

quen

ce(m

agen

ta).

The

yar

edi

rect

edto

the

Tic

/Toc

tran

sloc

atio

nco

mpl

exin

the

chlo

ropl

aste

nvel

ope

for

impo

rtin

toth

eor

gane

llein

anun

fold

edst

ate

(50)

.Fol

low

ing

impo

rt,t

heta

rget

ing

sequ

ence

sare

clea

ved

byth

est

rom

alSP

P.F

olde

dR

bcS

disp

lace

sass

embl

ych

aper

ones

(Raf

1an

d/or

Rbc

X)f

rom

Rbc

L8.

Fact

orsf

orm

etab

olic

repa

irof

Rub

isco

(sho

wn

inre

d)i

nclu

deR

caan

dsp

ecifi

csu

gar

phos

phat

ases

such

asX

uBP

ase.

Abb

revi

atio

ns:B

sd2,

bund

lesh

eath

defe

ctiv

e2;

CA

1P,2′ -c

arbo

xy-D

-ara

bini

tol-

1-ph

osph

ate;

CA

1Pas

e,C

A1P

phos

phat

ase;

PD

BP

, D-g

lyce

ro-2

,3-p

ento

diul

ose-

1,5-

bisp

hosp

hate

;2P

G,2

-pho

spho

glyc

olat

e;2P

Gas

e,2P

Gph

osph

atas

e;3P

GA

,3-p

hosp

hogl

ycer

ate;

Pi,

inor

gani

cph

osph

ate;

Raf

,Rub

isco

accu

mul

atio

nfa

ctor

;Rca

,Rub

isco

activ

ase;

Rub

isco

,rib

ulos

e-1,

5-bi

spho

spha

teca

rbox

ylas

e/ox

ygen

ase;

RuB

P,

ribu

lose

-1,5

-bis

phos

phat

e;SP

P,s

igna

lpep

tide

pept

idas

e;X

uBP

, D-x

ylul

ose-

1,5-

bisp

hosp

hate

;XuB

Pas

e,X

uBP

phos

phat

ase.

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As a further constraint, the extensive dependence of Rubisco on auxiliary factors for fold-ing, assembly, and maintenance may have affected its ability to evolve (45, 90) (Figure 10). Thechaperonin folding machinery promotes the structural evolution of proteins by buffering delete-rious effects of mutations on foldability and stability (10, 14, 45, 73, 126, 127, 142, 146), whereasthe specific assembly chaperone RbcX restricts the range of mutations compatible with Rubiscofunctionality (25). Moreover, RbcL-Raf1 complementarity is also required for holoenzyme as-sembly (139). Thus, RbcX and Raf1 interaction requirements would reinforce the evolutionarilytrapped state of Rubisco, with the compatibility needs of other assembly and maintenance factorslikely having a similar restrictive effect.

Strategies to Enhance Rubisco Function

In recent years, directed protein evolution has developed into a versatile tool for artificially mimick-ing and accelerating natural evolution (23). Directed evolution typically comprises the generationof a library of mutants followed by a selection process to identify mutant proteins with the desiredproperties. An engineered E. coli strain dependent on functional Rubisco for growth has beensuccessfully developed to screen libraries of Rubisco mutants (40, 80). So far, this approach hasyielded mainly mutants of Rubisco with increased solubility, but the potential to identify mutantswith improved catalytic properties has been demonstrated (40, 80), and such mutants have ex-hibited improved photosynthesis in cyanobacteria (25) and plants (143). However, only a minutefraction of the available mutational space has been sampled, especially when considering that mul-tiple mutations may be necessary to enhance Rubisco functionality (79, 140). Moreover, auxiliaryfactors may have to be coevolved to allow the expression of improved Rubisco variants.

Assuming that an improved Rubisco variant will be identified, the next challenge will be tointroduce it into a plant host in a way that allows functional expression and regulation. For thispurpose, plant genotypes tailored for bioengineering Rubisco have been developed and testedvia transformation of both the nucleus and chloroplast genomes (90, 140, 141). Such studieshave suggested that differences in the assembly requirements among Rubiscos influence theirproduction in leaf chloroplasts—explaining, for example, why efforts to express the catalyticallysuperior red-type Rubisco in green-type chloroplasts were not successful (138) and why only lowlevels of cyanobacterial RbcL8S8 are produced in tobacco chloroplasts (68, 85). Indeed, Whitneyet al. (139) recently showed that the biogenesis of heterologous Rubisco in tobacco leaves isdependent on the evolutionary compatibility between RbcL and the assembly factor Raf1. Bycontrast, the independence of auxiliary factors can explain why the bacterial RbcL2 is producedin tobacco leaves in amounts matching the levels of wild-type tobacco RbcL8S8 (143).

Other options to improve Rubisco performance in crop plants include the engineering ofmaintenance machinery (Rca and sugar phosphatases) and of CO2-concentrating mechanisms toreduce the energy cost associated with photorespiration (68, 71, 74, 87, 90, 94, 101, 124).

OUTLOOK

The necessity to meet the food demands of an increasing human population has reinvigoratedinterest in fundamental and applied research on Rubisco, and recent advances have providedexciting insights into the complex machineries used by cells to produce and maintain this abundantenzyme (Figure 10). A more complete understanding of the repertoire of factors dedicated toRubisco may hold promise for new bioengineering strategies based on rational design. Questions ofinterest include the unresolved mechanism of plant Rubisco assembly and functional maintenanceby its Rca. A better understanding of red-type Rubiscos from nongreen algae is of particular


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importance. The identification of auxiliary factors for these enzymes is lagging behind and hasbeen hampered by the lack of available genome sequences. It seems that Rubisco still holds manysecrets, and uncovering them will aid our capacity to modify its biogenesis and activity in a rangeof photosynthetic organisms—assisting efforts to improve agricultural yield for food and fuelproduction.

SUMMARY POINTS

1. Rubisco is the key enzyme of photosynthesis that catalyzes the fixation of atmosphericCO2 in the Calvin-Benson-Bassham cycle, converting inorganic to organic carbon andthus producing all biomass.

2. The most abundant form of Rubisco (form I) is a hexadecameric complex consisting ofeight large and eight small subunits.

3. Rubisco is remarkably inefficient as an enzyme and can make up nearly 50% of solubleleaf protein.

4. The biogenesis of Rubisco requires general chaperone machineries for subunit foldingand dedicated factors mediating assembly.

5. The activation and multistep catalytic reactions of Rubisco are error prone, producingsugar phosphate by-products that bind tightly to the active site and inhibit Rubiscofunction.

6. Metabolic repair of Rubisco requires remodeling of the enzyme by the AAA+ chaper-one Rubisco activase, releasing inhibitory sugar phosphates for hydrolysis by specificphosphatases.

7. Understanding the chaperone machineries for Rubisco biogenesis and functional main-tenance is important in efforts to engineer a better Rubisco, with the goal of improvingcrop yields.

DISCLOSURE STATEMENT

The authors are not aware of any affiliations, memberships, funding, or financial holdings thatmight be perceived as affecting the objectivity of this review.

ACKNOWLEDGMENTS

We thank G. Thieulin-Pardo and R. Wilson for critically reading the manuscript. The researchof M.H.-H. and S.M.W. is funded by the Minerva Foundation of the Max Planck Society and byAustralian Research Council grant CE14010001, respectively.

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PP68-FrontMatter ARI 10 March 2017 16:50

Annual Review ofPlant Biology

Volume 68, 2017 ContentsFirmly Planted, Always Moving

Natasha V. Raikhel � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 1

Biogenesis and Metabolic Maintenance of RubiscoAndreas Bracher, Spencer M. Whitney, F. Ulrich Hartl, and Manajit Hayer-Hartl � � � �29

The Epigenome and Transcriptional Dynamics of Fruit RipeningJames Giovannoni, Cuong Nguyen, Betsy Ampofo, Silin Zhong, and Zhangjun Fei � � � � � �61

Retrograde Signals: Integrators of Interorganellar Communication andOrchestrators of Plant DevelopmentAmancio de Souza, Jin-Zheng Wang, and Katayoon Dehesh � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �85

The Structural Basis of Ligand Perception and Signal Activation byReceptor KinasesUlrich Hohmann, Kelvin Lau, and Michael Hothorn � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 109

Cell Biology of the Plant NucleusIris Meier, Eric J. Richards, and David E. Evans � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 139

Phloem-Mobile RNAs as Systemic Signaling AgentsByung-Kook Ham and William J. Lucas � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 173

Chemical Genetic Dissection of Membrane TraffickingLorena Norambuena and Ricardo Tejos � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 197

Plant Mitochondrial Genomes: Dynamics and Mechanisms ofMutationJose M. Gualberto and Kathleen J. Newton � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 225

Plastoglobuli: Plastid Microcompartments with Integrated Functionsin Metabolism, Plastid Developmental Transitions, andEnvironmental AdaptationKlaas J. van Wijk and Felix Kessler � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 253

Strigolactone Signaling and EvolutionMark T. Waters, Caroline Gutjahr, Tom Bennett, and David C. Nelson � � � � � � � � � � � � � � � 291

Zooming In on Plant Hormone Analysis: Tissue- and Cell-SpecificApproachesOndrej Novak, Richard Napier, and Karin Ljung � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 323

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PP68-FrontMatter ARI 10 March 2017 16:50

Guilt by Association: A Phenotype-Based View of the PlantPhosphoinositide NetworkKatharina Gerth, Feng Lin, Wilhelm Menzel, Praveen Krishnamoorthy,

Irene Stenzel, Mareike Heilmann, and Ingo Heilmann � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 349

The Life and Death of a Plant CellMehdi Kabbage, Ryan Kessens, Lyric C. Bartholomay, and Brett Williams � � � � � � � � � � � � � 375

Genomics, Physiology, and Molecular Breeding Approaches forImproving Salt ToleranceAbdelbagi M. Ismail and Tomoaki Horie � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 405

New Strategies and Tools in Quantitative Genetics: How to Go fromthe Phenotype to the GenotypeChristos Bazakos, Mathieu Hanemian, Charlotte Trontin,

Jose M. Jimenez-Gomez, and Olivier Loudet � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 435

Novel Insights into Tree Biology and Genome Evolution as RevealedThrough GenomicsDavid B. Neale, Pedro J. Martınez-Garcıa, Amanda R. De La Torre,

Sara Montanari, and Xiao-Xin Wei � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 457

Defense Priming: An Adaptive Part of Induced ResistanceBrigitte Mauch-Mani, Ivan Baccelli, Estrella Luna, and Victor Flors � � � � � � � � � � � � � � � � � � � 485

Trade-Offs Between Plant Growth and Defense Against InsectHerbivory: An Emerging Mechanistic SynthesisTobias Zust and Anurag A. Agrawal � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 513

The Role of Plant Innate Immunity in the Legume-RhizobiumSymbiosisYangrong Cao, Morgan K. Halane, Walter Gassmann, and Gary Stacey � � � � � � � � � � � � � � 535

Plant Biodiversity Change Across Scales During the AnthropoceneMark Vellend, Lander Baeten, Antoine Becker-Scarpitta, Veronique Boucher-Lalonde,

Jenny L. McCune, Julie Messier, Isla H. Myers-Smith, and Dov F. Sax � � � � � � � � � � � � 563

Errata

An online log of corrections to Annual Review of Plant Biology articles may be found athttp://www.annualreviews.org/errata/arplant

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Documents

Biogenesis and Metabolic Maintenance of Rubisco