Embed Size (px)
Citation preview
Structural and Biochemical Elucidation of Mechanism forDecarboxylative Condensation of �-Keto Acid by CurcuminSynthase*□S
Received for publication, October 20, 2010, and in revised form, November 24, 2010 Published, JBC Papers in Press, December 9, 2010, DOI 10.1074/jbc.M110.196279
Yohei Katsuyama‡1, Ken-ichi Miyazono§, Masaru Tanokura§, Yasuo Ohnishi‡2, and Sueharu Horinouchi†‡
From the Departments of ‡Biotechnology and §Applied Biological Chemistry, Graduate School of Agriculture and Life Sciences,University of Tokyo, Bunkyo-ku, Tokyo 113-8657, Japan
The typical reaction catalyzed by type III polyketide syn-thases (PKSs) is a decarboxylative condensation between acyl-CoA (starter substrate) and malonyl-CoA (extender substrate).In contrast, curcumin synthase 1 (CURS1), which catalyzescurcumin synthesis by condensing feruloyl-CoA with adiketide-CoA, uses a �-keto acid (which is derived fromdiketide-CoA) as an extender substrate. Here, we determinedthe crystal structure of CURS1 at 2.32 A resolution. The over-all structure of CURS1 was very similar to the reported struc-tures of type III PKSs and exhibited the ����� fold. However,CURS1 had a unique hydrophobic cavity in the CoA-bindingtunnel. Replacement of Gly-211 with Phe greatly reduced theenzyme activity. The crystal structure of the G211F mutant (at2.5 A resolution) revealed that the side chain of Phe-211 occu-pied the hydrophobic cavity. Biochemical studies demon-strated that CURS1 catalyzes the decarboxylative condensa-tion of a �-keto acid using a mechanism identical to that fornormal decarboxylative condensation of malonyl-CoA by typi-cal type III PKSs. Furthermore, the extender substrate speci-ficity of CURS1 suggested that hydrophobic interaction be-tween CURS1 and a �-keto acid may be important for CURS1to use an extender substrate lacking the CoA moiety. Fromthese results and a modeling study on substrate binding, weconcluded that the hydrophobic cavity is responsible for thehydrophobic interaction between CURS1 and a �-keto acid,and this hydrophobic interaction enables the �-keto acid moi-ety to access the catalytic center of CURS1 efficiently.
Type III polyketide synthases (PKSs)3 are distributed indiverse organisms, including plants, fungi, and bacteria, and
are responsible for the syntheses of various biologically andpharmaceutically important compounds (1, 2). The universalreactions catalyzed by type III PKSs are as follows: (i) transferof acyl-CoA (called the starter substrate) to the catalytic Cys,resulting in an acyl-PKS complex; (ii) decarboxylation of mal-onyl-CoA (called the extender substrate) to form an activeanion; (iii) Claisen condensation of the active anion with theacyl moiety attached to the catalytic Cys to generate an acyl-CoA that has an additional two-carbon unit; (iv) extension ofthe polyketide chain by repeating reactions i–iii; and (v) cycli-zation of the resultant polyketide chain and release from theenzyme (1, 2). As an example, the reaction catalyzed by chal-cone synthase (CHS), which is a type III PKS, is depicted inFig. 1A (1, 2). The most important amino acid residues fortype III PKSs are Cys-164, His-303, and Asn-336 (numberingin CHS), which create a catalytic triad. Cys-164 in CHS formsa thioester bond with p-coumaroyl-CoA (starter substrate)and a polyketide intermediate. His-303 and Asn-336 are re-sponsible for acyl-PKS complex formation and the decarboxy-lative condensation of malonyl-CoA (1, 2). Type III PKSsform homodimers, and each monomer has a characteristicstructure, called the ����� fold (3–7). The catalytic triad liesin the middle of each molecule and is connected to the sur-face by a CoA-binding tunnel, so-called because cocrystalliza-tion of CoA with several type III PKSs revealed that the CoAmolecule bound to this tunnel (4–7). Thus, this tunnel proba-bly enables an acyl moiety of acyl-CoA (or malonyl-CoA) toaccess the catalytic center efficiently (4, 5). On the oppositeside of the CoA-binding tunnel, a cavity exists that accommo-dates the growing polyketide chain and facilitates cyclizationof the polyketide chain (4–7). The diverse polyketides pro-duced by type III PKSs arise from the specificity for starterand extender substrates, the number of incorporated extendersubstrates, and the cyclization pattern of the polyketideintermediate.Until recently, the extender substrate used by type III PKSs
was believed to be limited to malonyl-CoA and its derivativeCoAs (e.g.methylmalonyl-CoA). However, we recently dis-covered a novel class of type III PKSs that uses a �-keto acidas an extender substrate; this class catalyzes the decarboxyla-tive condensation between an acyl-CoA and a �-keto acid,resulting in unusual head-to-head condensation of polyketidechains (8–11). Curcumin synthases 1–3 (CURS1, CURS2, andCURS3) (9, 10) from Curcuma longa catalyze formation ofcurcumin by condensing feruloyl-CoA with the �-keto acid,
* This work was supported in part by Targeted Proteins Research Programof the Ministry of Education, Culture, Sports, Science, and Technology ofJapan.
□S The on-line version of this article (available at http://www.jbc.org) con-tains supplemental Figs. S1–S6, Tables S1 and S2, and an additionalreference.
The atomic coordinates and structure factors (codes 3OV2 and 3OV3) havebeen deposited in the Protein Data Bank, Research Collaboratory forStructural Bioinformatics, Rutgers University, New Brunswick, NJ(http://www.rcsb.org/).
† Deceased July 12, 2009.1 Supported by the Japan Society for the Promotion of Science.2 To whom correspondence should be addressed. Tel.: 81-3-58415123; Fax:
81-3-58418021; E-mail: [email protected] The abbreviations used are: PKS, polyketide synthase; BAS, benzalacetone
synthase; CHS, chalcone synthase; CURS, curcumin synthase; NAC,N-acetylcysteamine.
THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 286, NO. 8, pp. 6659 –6668, February 25, 2011© 2011 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.
FEBRUARY 25, 2011 • VOLUME 286 • NUMBER 8 JOURNAL OF BIOLOGICAL CHEMISTRY 6659
by guest on March 6, 2020
http://ww
w.jbc.org/
Dow
nloaded from
5-(4-hydroxy-3-methoxyphenyl)-3-oxo-4-pentenoic acid.Curcuminoid synthase from Oryza sativa catalyzes the invitro formation of bisdemethoxycurcumin by condensing p-coumaroyl-CoA with the �-keto acid, 5-(4-hydroxyphenyl)-3-oxo-4-pentenoic acid. In addition to these enzymes, ginger,banana, and red root (Wachendorfia thyrsiflora) have beenbelieved to possess some type III PKSs of this class (12–14).Furthermore, PqsD, a 3-oxoketoacyl-acyl carrier protein syn-thase III (KASIII) related to type III PKSs, is predicted to cata-lyze the condensation between anthraniloyl-CoA and a 3-oxofatty acid in 4-hydroxy-2-heptylquinoline synthesis in Pseudo-monas aeruginosa (15–17). This reaction may be catalyzed bya mechanism similar to that of curcuminoid synthase and cur-cumin synthases (15). A KASIII involved in the biosynthesisof 3,6,9,12,15,19,22,25,28-hentriacontanonaene in Shewanellaoneidensis strain MR-1 also appeared to catalyze this similarreaction (18). Thus, the unusual head-to-head condensationof polyketide chains occurs not only in plants, but also in mi-croorganisms, and it can be catalyzed not only by type IIIPKSs but also by other types of ketosynthases.CURS1 catalyzes two reactions. First, CURS1 catalyzes hy-
drolysis of feruloyldiketide-CoA, which is synthesized bydiketide-CoA synthase (Fig. 1B, i), to form 5-(4-hydroxy-3-methoxyphenyl)-3-oxo-4-pentenoic acid (Fig. 1B, ii) (9). Sec-ond, using a �-keto acid as an extender substrate, CURS1 cat-alyzes condensation of the �-keto acid with feruloyl-CoA(starter substrate) to form curcumin (Fig. 1B, iii). Because no�-keto acid was detected in the in vitro CURS1 reaction, the�-keto acid was apparently not released from the enzyme (4).Thus, condensation of the resultant �-keto acid with feruloyl-CoA is assumed to occur immediately after hydrolysis of feru-
loyldiketide-CoA in the active site pocket. Notably, CURS1rarely catalyzes normal head-to-tail condensation betweenferuloyl-CoA (starter substrate) and malonyl-CoA (extendersubstrate; Fig. 1B, i). Instead, this reaction is apparently cata-lyzed by diketide-CoA synthase in C. longa as described above(4). Because acyl-CoA hydrolysis activity has been reportedfor several other type III PKSs, including benzalacetone syn-thase (BAS) (6, 19), the most intriguing characteristic ofCURS1 is its ability to use a �-keto acid as an extender sub-strate. However, the only direct evidence that supports thismechanism is detection of condensation activity betweenferuloyl-CoA and a �-keto acid in vitro. Thus, further experi-ments are required to clarify how CURS1 catalyzes condensationbetween feruloyl-CoA and diketide-CoA. Two questions areraised regarding condensation between feruloyl-CoA and a�-keto acid. First, does CURS1 catalyze the decarboxylative con-densation of a �-keto acid using the identical mechanism as thatfor the normal decarboxylative condensation of malonyl-CoA bytypical type III PKSs? Second, how does CURS1 bind the ex-tender substrate that lacks a CoAmoiety? To address these ques-tions, we determined the crystal structure of CURS1 at 2.32 Åresolution, followed by site-directedmutagenesis, biochemicalexperiments, and amodeling study.We succeeded in providingthe first structural insights into themolecular mechanism for theunique reaction catalyzed by CURS1.
EXPERIMENTAL PROCEDURES
Materials—Escherichia coli strains JM109 and BLR(DE3),plasmid pUC19, restriction enzymes, T4 DNA ligase, TaqDNA polymerase, PrimeSTAR HS DNA polymerase, andother DNA-modifying enzymes were purchased from Takara
FIGURE 1. Reactions catalyzed by chalcone synthase (A) and by curcumin synthase 1 (B and C). A, CHS catalyzes condensation of p-coumaroyl-CoA andthree molecules of malonyl-CoA to synthesize the tetraketide intermediate. The resulting tetraketide intermediate is further cyclized by CHS and convertedto naingenin chalcone. B, CURS1 catalyzes the hydrolysis of diketide-CoA to yield a �-keto acid (ii) and decarboxylative condensation of the �-keto acid withferuloyl-CoA to yield curcumin (iii). CURS1 scarcely catalyzes the formation of diketide-CoA from feruloyl-CoA and malonyl-CoA (i). In C. longa, this reactionis primarily catalyzed by a diketide-CoA synthase. C, for analysis of the activity of CURS1, cinnamoylferuloylmethane (curcuminoid) formation from feruloyl-CoA with cinnamoyldiketide-NAC (an analog of diketide-CoA) or 3-oxo-5-phenyl-4-pentenoic acid (a �-keto acid) was examined.
Decarboxylative Condensation of �-Keto Acid by CURS1
6660 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 286 • NUMBER 8 • FEBRUARY 25, 2011
by guest on March 6, 2020
http://ww
w.jbc.org/
Dow
nloaded from
Biochemicals (Shiga, Japan). Plasmids pET16b and pET26bwere purchased from Novagen (Darmstadt, Germany). Ferulicacid and curcumin were purchased fromWako Chemicals(Osaka, Japan). Cinnamoylferuloylmethane, cinnamoyl-diketide N-acetylcysteamine (NAC), dihydrocinnamoyl-diketide-NAC, 3-oxooctanoyl-NAC, and 3-oxopalmitoyl-NAC were synthesized as described previously (8, 9). PlasmidpET16b-CURS1 was described previously (9). 3-Oxo-5-phe-nyl-4-pentenoic acid methyl ester was synthesized as de-scribed previously (8, 9). Acetoacetic acid and 3-oxovalericacid methyl ethers were purchased from Tokyo Chemical In-dustry (Tokyo, Japan). To avoid the nonenzymatic decarboxy-lation of 3-oxo-5-phenyl-4-pentenoic acid, acetoacetic acid,and 3-oxovaleric acid, the acids were prepared by alkalinehydrolysis of 3-oxo-5-phenyl-4-pentenoic acid, acetoaceticacid, and 3-oxovaleric acid methyl esters immediately beforeuse.Construction of pET26b-CURS1—Using pET16b-CURS1 (9)
as a template, the DNA fragment encoding curcumin syn-thase was amplified by PCR with primer 5�-CCGAATTC-CATATGGCCAACCTCCACGCGTT-3� (underlining indi-cates the NdeI site, boldface letters indicate the EcoRI site,and italic letters indicate the start codon of the CURS1 gene)and 5�-CGCGGATCCTCAGTGGTGGTGGTGGT-TCAGTCTGCAACTATGGA-3� (underlining indicates theBamHI site, boldface letters indicate four codons for His resi-dues fused to the C terminus of CURS1, and italic letters indi-cate an artificially generated stop codon). Amplified fragmentwas cloned between EcoRI and BamHI sites of pUC19, result-ing in pUC19-CURS1-His4. pUC19-CURS1-His4 was digestedwith NdeI and BamHI, and the DNA fragment encodingCURS1 with four His was cloned between NdeI and BamHIsites of pET26b, resulting in pET26b-CURS1-His4. An expres-sion plasmid (pET26b-CURS1 (G211F)-His4) for productionof CURS1 (G211F) with four His residues at its C terminuswas constructed by replacing the 954-bp NdeI-PstI fragmentof pET26b-CUSRS1-His4 with that of pET16b-CURS1(G211F) (see below).Production, Purification, and Crystallization of CURS1-His4—
CURS1 with four His residues at its C terminus (CURS1-His4)was produced and purified as described below. The E. coliBL21(DE3) strain harboring pET26b-CURS1-His4 was inocu-lated into 10 ml of Luria Bertani (LB) medium containing 50mg/liter kanamycin and incubated at 37 °C overnight. A por-tion (7 ml) of the preculture was inoculated into 3 liters of LBmedium containing a few drops of antifoam SI (Wako Chemi-cals) and 50 mg/liter kanamycin and incubated at 37 °C for2.5 h. The culture broth was cooled to 26 °C; isopropyl �-D-1-thiogalactopyranoside was added at a final concentration of0.5 mM, and the culture was further incubated at 26 °C over-night. The cells were harvested by centrifugation, then resus-pended in a buffer containing 20 mM Tris-HCl (pH 8.0), 10%glycerol, and 1 mM dithiothreitol, and disrupted by sonication.The cell debris was removed by centrifugation and filtration.CURS1-His4 in the solution was purified using nickel-nitrilotriacetic acid Superflow resin (Qiagen, Hilden, Ger-many) according to the manufacturer’s instructions. Imida-zole was removed by dialysis, and CURS1-His4 was further
purified by an anion exchange column Resource Q (GEHealthcare) using AKTAprime (GE Healthcare). The solutioncontaining purified CURS1-His4 was exchanged with a buffercontaining 10 mM Tris-HCl (pH 8.0), 10% glycerol, and 1 mM
tris(2-carboxyethyl)phosphine by ultrafiltration. The solutioncontaining 10 mg/ml CURS1 was used for crystallization.CURS1-His4 was crystallized using the sitting drop vapor dif-fusion method with a reservoir containing 0.3 M sodium mal-onate (pH 7.0) and 25% PEG3350. X-ray data were collectedusing beamline AR-NW12A (Photon Factory, Tsukuba, Ja-pan). The phases were determined using the molecular re-placement program (MOLREP) (20) with the crystal structureof CHS (1BQ6). Model building and refinement were per-formed using the Coot (21) and Refmac5 (22) programs. Simi-larly, CURS1 (G211F) with four His residues at its C terminuswas crystallized, and x-ray data were collected. The phaseswere determined using the MOLREP program with the crystalstructure of the wild-type CURS1. The details of the data col-lection and refinement statistics are summarized in supple-mental Table S1. The coordinates of CURS1 and CURS1(G211F) have been deposited into the Protein Data Bank withthe accession numbers 3OV2 and 3OV3, respectively.Construction of Expression Plasmids for Production of
CURS1 Mutants—Linearized pET16b-CURS1 plasmids withmutations were amplified by PCR with primers in supplemen-tal Table S2 using pET16b-CURS1 as a template. The tem-plate pET16b-CURS1 was digested with DpnI. LinearizedpET16b-CURS1 plasmids containing mutations were phos-phorylated using T4 polynucleotide kinase and ligated to cir-cular plasmids using T4 DNA ligase. E. coli JM109 was trans-formed using each of the plasmids, and mutations wereconfirmed by nucleotide sequencing. The recombinantCURS1 proteins were produced and purified as described pre-viously (9).Enzyme Assay—The standard reaction mixture contained
100 �M feruloyl-CoA, 100 �M extender substrate (cin-namoyldiketide-NAC, or 3-oxo-5-phenyl-4-pentenoic acid),100 mM potassium phosphate buffer (pH 8.0), and 4.0 �g ofrecombinant CURS1 protein in a total volume of 100 �l. Re-actions were incubated at 37 °C for 20 min (cinnamoyl-diketide-NAC), 5 min (3-oxo-5-phenyl-4-pentenoic acid), and1 h (others) before being quenched with 20 �l of 6 M HCl. Theproducts were extracted with ethyl acetate, and the organiclayer was evaporated to dryness. The residual materials weredissolved in 20 �l of dimethyl sulfoxide for HPLC analysis.The HPLC instrument (Waters) was equipped with a Doco-sil-B reversed-phase HPLC column (4.6 � 250 mm; SenshuScientific, Tokyo, Japan) and eluted using a linear acetonitrilegradient (10–100% over 30 min) in water containing 0.1%acetic acid at flow rate of 1.0 ml/min.Kinetic Analysis of CURS1 and CURS1 Mutants—The reac-
tion mixture contained 100 mM potassium phosphate buffer(pH 7.5), 1–100 �M each of feruloyl-CoA with 200 �M cin-namoyldiketide-NAC or 100 �M 3-oxo-5-phenyl-4-pentenoicacid, and 0.2–4.0 �g of recombinant CURS1 protein in a totalvolume of 100 �l. After the reaction mixture without sub-strates was preincubated at 37 °C for 2 min, the reaction wasinitiated by adding the substrates and was then continued for
Decarboxylative Condensation of �-Keto Acid by CURS1
FEBRUARY 25, 2011 • VOLUME 286 • NUMBER 8 JOURNAL OF BIOLOGICAL CHEMISTRY 6661
by guest on March 6, 2020
http://ww
w.jbc.org/
Dow
nloaded from
2.5–5 min. Next, the reaction was quenched by adding 20 �lof 6 M HCl. The solution was extracted with ethyl acetate, andthe organic layer was evaporated to dryness. Finally, the resid-ual materials were dissolved in 40 �l of dimethyl sulfoxide forHPLC analysis. Authentic cinnamoylferuloylmethane wasused to generate standard curves for quantification of theproducts. Steady-state parameters were determined by fittingto the equation, v � [S]Vmax/([S] � Km).
RESULTS
Overall Structure of CURS1—We determined the crystalstructure of recombinant CURS1-His4 at 2.32 Å resolution.A unit cell of the CURS1 crystal contained four moleculesof CURS1, two dimers. All the monomers are highly similar
to each other, with root mean square deviations of 0.4. Theoverall structure of the CURS1 dimer was very similar tothe structures of some type III PKSs and exhibited the����� fold (Fig. 2, A and B) (1, 2, 4). Met-137 of eachmonomer was located adjacent to the active site pocket ofthe other monomer and created a wall beside the active sitepocket, as reported for other type III PKSs (Fig. 2A) (4, 6,19). The catalytic triad (Cys-164, His-303, and Asn-336)was buried in the middle of each monomer and was con-nected to the surface with a CoA-binding tunnel. The twocatalytic centers of a dimer were apparently inaccessible toeach other because no tunnel connected them. Thus, twoactive sites can work independently of each other, similarto other type III PKSs.
A
His-303
Asn-336
Cys-164
CoA-bindingtunnel
ClCURS1 1 ----------MANLHALRREQRAQGPATIMAIGTATPPNLYEQSTFPDFYFRVTNSDDKQELKKKFRRMCEKTMVKKRYLHLTEEILKERPKLCSOsCUS 1 MAPTTTMGSALYPLGEMRRSQRADGLAAVLAIGTANPPNCVTQEEIPDFYFRVTNSDHLTALKDKFKRICQEMGVQRRYLHHTEEMLSAHPEFVDClDCS 1 -----------MEANGYRITHSADGPATILAIGTANPTNVVDQNAYPDFYFRVTNSEYLQELKAKFRRICEKAAIRKRHLYLTEEILRENPSLLAMsCHS 1 ----------MVSVSEIRKAQRAEGPATILAIGTANPANCVEQSTYPDFYFKITNSEHKTELKEKFQRMCDKSMIKRRYMYLTEEILKENPNVCERhBAS 1 -----------------MATEEMKKLATVMAIGTANPPNCYYQADFPDFYFRVTNSDHLINLKQKFKRLCENSRIEKRYLHVTEEILKENPNIAA
ClCURS1 86 YKEASFDDRQDIVVEEIPRLAKEAAEKAIKEWGRPKSEITHLVFCSISGIDMPGADYRLATLLGLPLTVNRLMIYSQACHMGAAMLRIAKDLAENOsCUS 96 RDAPSLDARLDIAADAVPELAAEAAKKAIAEWGRPAADITHLVVTTNSGAHVPGVDFRLVPLLGLRPSVRRTMLHLNGCFAGCAALRLAKDLAENClDCS 85 PMAPSFDARQAIVVEAVPKLAKEAAEKAIKEWGRPKSDITHLVFCSASGIDMPGSDLQLLKLLGLPPSVNRVMLYNVGCHAGGTALRVAKDLAENMsCHS 86 YMAPSLDARQDMVVVEVPRLGKEAAVKAIKEWGQPKSKITHLIVCTTSGVDMPGADYQLTKLLGLRPYVKRYMMYQQGCFAGGTVLRLAKDLAENRhBAS 79 YEATSLNVRHKMQVKGVAELGKEAALKAIKEWGQPKSKITHLIVCCLAGVDMPGADYQLTKLLDLDPSVKRFMFYHLGCYAGGTVLRLAKDIAEN
ClCURS1 181 NRGARVLVVACEITVLSFRGPNEGDFEALAGQAGFGDGAGAVVVGADPLEGIEKPIYEIAAAMQETVAESQGAVGGHLRAFGWTFYFLNQLPAIIOsCUS 191 SRGARVLVVAAELTLMYFTGPDEGCFRTLLVQGLFGDGAAAVIVGAD-ADDVERPLFEIVSAAQTIIPESDHALNMRFTERRLDGVLGRQVPGLIClDCS 180 NRGARVLAVCSEVTVLSYRGPHPAHIESLFVQALFGDGAAALVVGSDPVDGVERPIFEIASASQVMLPESAEAVGGHLREIGLTFHLKSQLPSIIMsCHS 181 NKGARVLVVCSEVTAVTFRGPSDTHLDSLVGQALFGDGAAALIVGSDPVPEIEKPIFEMVWTAQTIAPDSEGAIDGHLREAGLTFHLLKDVPGIVRhBAS 174 NKGARVLIVCSEMTTTCFRGPSETHLDSMIGQAILGDGAAAVIVGADPDLTVERPIFELVSTAQTIVPESHGAIEGHLLESGLSFHLYKTVPTLI
ClCURS1 276 ADNLGRSLERALAPLG----VREWNDVFWVAHPGNWAIIDAIEAKLQLSPDKLSTARHVFTEYGNMQSATVYFVMDELRKRSAVEGRSTTGDGLQOsCUS 285 GDNVERCLLDMFGPLLGGDGGGGWNDLFWAVHPGSSTIMDQVDAALGLEPGKLAASRRVLSDYGNMSGATVIFALDELRRQRKE--AAAAGEWPEClDCS 275 ASNIEQSLTTACSPLG----LSDWNQLFWAVHPGGRAILDQVEARLGLEKDRLAATRHVLSEYGNMQSATVLFILDEMRNRSAAEGHATTGEGLDMsCHS 276 SKNITKALVEAFEPLG----ISDYNSIFWIAHPGGPAILDQVEQKLALKPEKMNATREVLSEYGNMSSACVLFILDEMRKKSTQNGLKTTGEGLERhBAS 269 SNNIKTCLSDAFTPLN ISDWNSLFWIAHPGGPAILDQVTAKVGLEKEKLKVTRQVLKDYGNMSSATVFFIMDEMRKKSLENGQATTGEGLE
B
C
CoA boundwith CHS
----
Met-137
FIGURE 2. Overall structure of CURS1 (A and B) and amino acid alignment of CURS1 with other type III PKSs (C). A, CURS1 dimer exhibited the �����fold, similar to other type III PKSs. One monomer is highlighted in green and the other in dark blue. The side chains of Cys-164, His-303, Asn-336, and Met-137 are depicted. B, overall structure of a CURS1 monomer (green) is superimposed on that of CHS (magenta). Both enzymes have a very similar structure.C, catalytic triad (Cys-164, His-303, and Asn-336 in CURS1) is highlighted in orange and two phenylalanines (Phe-215 and Phe-265 in CURS1) that are called“gatekeepers” are highlighted in blue. ClCURS1, CURS1 (curcumin synthase 1) from C. longa (BAH56226); OsCUS, curcuminoid synthase from O. sativa(AK109558); ClDCS, diketide-CoA synthase from C. longa (BAH56225); MsCHS, CHS from Medicago sativa (AAA02824); RhBAS, BAS from Rheum palmatum(AAK82824).
Decarboxylative Condensation of �-Keto Acid by CURS1
6662 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 286 • NUMBER 8 • FEBRUARY 25, 2011
by guest on March 6, 2020
http://ww
w.jbc.org/
Dow
nloaded from
Characteristics of the Active Site of CURS1—Similar toother type III PKSs, CURS1 had an active site pocket com-posed of three parts as follows: (i) a CoA-binding tunnel thatrepresents the entrance for the substrates; (ii) the catalytictriad; and (iii) a cavity that could accommodate an extendedpolyketide chain (1–5). In addition to the catalytic triad (Cys-164, His-303, and Asn-336), two well conserved Phe residues,Phe-215 and Phe-265, were retained by CURS1 (Fig. 2C).These two Phe residues are called the gatekeepers (1, 2). How-ever, the orientation of the Phe-265 side chain was differentfrom that of the corresponding Phe residues (Phe-265) ofCHS (Fig. 3B) (4). This difference resulted in a slightly widerCoA-binding tunnel of CURS1 in comparison with CHS.More importantly, the Phe-265 side chain of CURS1 provideda hydrophobic cavity in the CoA-binding tunnel. The hydro-phobic cavity was surrounded by the side chains of Phe-265,Phe-215, Phe-267, and Gly-211 (Fig. 3A). In contrast, a cavitythat would accommodate an extended polyketide chain inCURS1 was much narrower than that of CHS because of thesubstitution of Ser-338 (in CHS) by Gln-338 and the change
in orientation of the Phe-265 side chain (Fig. 3B). Unexpect-edly, near the catalytic Cys of each monomer, electron densityexisted that was probably derived from malonic acid, whichwas included in the crystallization buffer (Fig. 3 and supple-mental Fig. S1). Although the orientation of malonic acid wassomewhat different from monomer to monomer (supplemen-tal Fig. S1E), malonic acid formed hydrogen bonds with thecatalytic residues His-303 and Asn-336 in every monomer.This observation suggested that malonic acid could mimic the�-keto acid moiety of the extender �-keto acid. Malonic acidin chain A was depicted in Fig. 3, because its electron densitywas most clear among four malonic acid molecules includedin the crystal structure (one each monomer) and because po-sition of the molecule appeared to be significant as a �-ketoacid analog; the center carbon of malonic acid should be lo-cated sufficiently close to the catalytic Cys.Role of the Catalytic Triad in Decarboxylative Condensation
of a �-Keto Acid and Starter Substrate Loading—To investi-gate the functional role of His-303 in �-keto acid condensa-tion by CURS1, active site mutant enzymes (H303Q and
A
B
lle-275
Leu-377
Phe-373
His-303
Cys-164Ala-163
Met-137
Ile-132
Gln-338
Phe-265
Phe-215
Gly-211Ala-210
Phe-267
Asn-336
Val-254
Ile-309
Leu-271
Asn-306
Thr-194
Ser-197
lle-275
Leu-377
Phe-373
His-303
Cys-164Ala-163
Met-137
Ile-132
Gln-338
Phe-265
Phe-215
Gly-211Ala-210
Phe-267
Asn-336
Val-254
Ile-309
Leu-271
Asn-306
Thr-194
Ser-197
FIGURE 3. Wall-eye stereo view of the structure of active site pocket of CURS1 (chain A). A, white sticks indicate the side chains of the amino acid resi-dues around the active site pocket of CURS1. The malonic acid observed in the active site pocket is shown as green sticks. The cyan sticks indicate the CoA(left) and naringenin (right) bound with CHS. They are superimposed with CURS1 for the comparison of the active site pockets between CURS1 and CHS.B, side chains of the amino acid residues around the active site pocket of CHS (orange) are superimposed on those of CURS1 (white).
Decarboxylative Condensation of �-Keto Acid by CURS1
FEBRUARY 25, 2011 • VOLUME 286 • NUMBER 8 JOURNAL OF BIOLOGICAL CHEMISTRY 6663
by guest on March 6, 2020
http://ww
w.jbc.org/
Dow
nloaded from
H303A) were prepared. The curcuminoid synthesis activity ofCURS1 was examined using two different enzyme assays. Oneinvolved curcuminoid synthesis from feruloyl-CoA and cin-namoyldiketide-NAC (Fig. 1C, ii and iii). The other was thatfrom feruloyl-CoA and 3-oxo-5-phenyl-4-pentenoic acid (Fig.1C, iii). The former included two reactions as follows: hydrol-ysis of cinnamoyldiketide-NAC and condensation of the re-sultant �-keto acid with feruloyl-CoA. In contrast, the latterincluded only condensation of the �-keto acid with feruloyl-CoA, allowing us to examine the direct influence of these mu-tations on �-keto acid condensation. Notably, we used cin-namoyldiketide-NAC and 3-oxo-5-phenyl-4-pentenoic acidas analogs of feruloyldiketide-CoA and 5-(4-hydroxy-3-me-thoxyphenyl)-3-oxo-4-pentenoic acid, respectively, because ofthe ease of their chemical synthesis. When either of the mu-tant CURS1 enzymes (CURS1 H303Q and CURS1 H303A)was used, the yield of the curcuminoid product decreasedmarkedly in both assays (Fig. 4). This result suggested thatHis-303 is important for �-keto acid condensation. Notably,the corresponding CHS mutants (CHS H303Q and CHSH303A) similarly showed a marked decrease in the decar-boxylative condensation of malonyl-CoA (23, 24).We then determined the kinetic parameters of CURS1
H303Q and wild-type CURS1 in curcuminoid formation fromferuloyl-CoA and cinnamoyldiketide-NAC, as well as thatfrom feruloyl-CoA and 3-oxo-5-phenyl-4-pentenoic acid
(Table 1). The kcat of curcuminoid formation from 3-oxo-5-phenyl-4-pentenoic acid was much higher than that fromcinnamoyldiketide-NAC. This result indicated that the hy-drolysis step is rate-limiting in curcuminoid formation fromferuloyl-CoA and diketide-CoA, excluding the possibility thatcondensation precedes hydrolysis in the reaction. In theH303Q mutant, curcuminoid synthesis activity from 3-oxo-5-phenyl-4-pentenoic acid decreased �10-fold in comparisonwith the wild-type enzyme. Decreased curcuminoid synthesisactivity resulted from a marked reduction of the kcat value.Introduction of the H303Q mutation into CHS similarly al-tered its enzymatic property (24), suggesting that the catalyticHis residue has the same function between CURS1 and CHSin the decarboxylative condensation of the �-keto acid andmalonyl-CoA, respectively.In CHS, His-303 is involved not only in the decarboxylative
condensation of malonyl-CoA but also in starter substrateloading (23, 24). To obtain further insight into the role of His-303, we examined the dependence of the enzyme activity onpH in curcuminoid synthesis from feruloyl-CoA and 3-oxo-5-phenyl-4-pentenoic acid using the H303Q mutant and thewild-type enzyme (supplemental Fig. S2). In the wild-typeenzyme, the optimum pH was 7.0, and almost no reduction inthe activity occurred between pH 6.0 and 7.0. In contrast, inthe H303Q mutant, the optimum pH was 7.5, and the activitygradually decreased when the pH was lowered. This differ-ence could be explained by the role of His-303 in the startersubstrate loading, which was described in analysis of the reac-tion mechanism of CHS (23, 24). In CHS, His-303 acts as thegeneral base that takes the proton from the thiol moiety ofCys-164, which lowers the pKa of the thiol moiety (23, 24).The deprotonated thiol residue of Cys-164 reacts with astarter substrate to facilitate starter substrate loading. Thereplacement of His-303 by Gln results in an increase in thepKa of Cys-164, which raises the optimum pH of the startersubstrate loading and decreases the activity under lower pHconditions (23, 24). Thus, this result indicated that His-303 inCURS1 may function similarly to the His-303 residue of CHS,not only in decarboxylative condensation of a �-keto acid butalso in starter substrate loading.Thus, all of these results supported the idea that CURS1
catalyzes decarboxylative condensation of a �-keto acid usingthe same mechanism as that for normal decarboxylative con-densation of malonyl-CoA by typical type III PKSs. This ideawas also supported by the following observations. First, therecombinant CURS1 protein demonstrated weak decarboxy-lation activity toward 3-oxo-5-phenyl-4-pentenoic acid (sup-plemental Fig. S3), similar to typical type III PKSs that havedecarboxylation activity toward malonyl-CoA (23, 24). Sec-
FIGURE 4. Curcuminoid synthesis activity of the wild-type and mutantCURS1 enzymes from feruloyl-CoA with cinnamoyldiketide-NAC (graybars) or 3-oxo-5-phenyl-4-pentenoic acid (�-keto acid) (white bars).
TABLE 1Kinetic parameters of recombinant CURS1 proteins in curcuminoid synthesis from feruloyl-CoA and cinnamoyldiketide-NAC or 3-oxo-5-phenyl-4-pentenoic acid (n � 3)
Cinnamoyldiketide-NAC 3-Oxo-5-phenyl-4-pentenoic acidWT G211F H303Q WT G211F H303Q
kcat (1/min) 0.26 � 0.01 0.088 � 0.010 0.0044 � 0.0002 7.5 � 2.7 0.50 � 0.09 0.10 � 0.01Km (�M)a 1.7 � 0.5 113 � 6 3.0 � 0.0 5.9 � 1.7 138 � 88 1.2 � 0.3kcat/Km (1/s/M) 2915 � 650 13 � 1 24 � 1 20541 � 3487 112 � 44 1605 � 456
aKm values for feruloyl-CoA are shown.
Decarboxylative Condensation of �-Keto Acid by CURS1
6664 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 286 • NUMBER 8 • FEBRUARY 25, 2011
by guest on March 6, 2020
http://ww
w.jbc.org/
Dow
nloaded from
ond, generation of a covalent bond between CURS1 and feru-loyl-CoA was observed (supplemental Fig. S4). Third, theC164S mutation abolished the activity to synthesize curcumi-noid from feruloyl-CoA and cinnamoyldiketide-NAC or a�-keto acid (data not shown). These results showed that thecovalent bond between Cys-164 and the feruloyl moiety wasnecessary for �-keto acid condensation.Extender Substrate Specificity of CURS1—To gain insight
into the unique ability of CURS1 to use a �-keto acid as anextender substrate, we analyzed the extender substrate speci-ficity of CURS1. CURS1 was incubated with several diketide-NAC (or -CoA) molecules or �-keto acids in the presence offeruloyl-CoA as a starter substrate. CURS1 used dihydrocin-namoyldiketide-NAC, 3-oxooctanoyl-NAC, and 3-oxopalmi-toyl-NAC as extender substrates and produced curcuminoidderivatives (see supplemental Fig. S5 for their structures), sug-gesting that CURS1 has a relatively relaxed extender substratespecificity toward diketide-CoAs with a hydrophobic moiety.However, when CURS1 was incubated with acetoacetyl-CoA,acetoacetic acid, or 3-oxovaleric acid, no curcuminoid deriva-tive was detected. This suggested that diketide-CoAs (�-ketoacids) derived from short chain fatty acids were not used byCURS1. We assumed that hydrophobic interaction betweenCURS1 and a �-keto acid would be important, and thus a hy-drophobic moiety (a phenyl group or a long alkyl chain) of a�-keto acid would be essential for the �-keto acid to be usedas an extender substrate of CURS1.Involvement of the Hydrophobic Cavity around Phe-265 in
�-Keto Acid Condensation—All structurally characterizedtype III PKSs have a CoA-binding tunnel that serves as theentrance for the starter and extender substrates, and severalamino acid residues in the tunnel are responsible for bindingof the CoA moiety of the substrates (1, 2). Because CURS1can use an extender substrate that lacks the CoA moiety, itmay have an alternative mechanism for binding the extendersubstrate. As described above, we assumed that the hydro-phobic interaction between CURS1 and a �-keto acid wouldbe important. The hydrophobic cavity around Phe-265 in theCoA-binding tunnel was very likely to be involved in the hy-drophobic interaction. Thus, we constructed two mutantCURS1 proteins, in which Gly-211 was replaced by the bulk-ier amino acids, Phe and Trp. Because Gly-211 is located inthe hydrophobic cavity, an aromatic group of the side chain ofthe Phe-211 and Trp-211 of each of the mutant enzymes wasassumed to occupy this hydrophobic cavity.As expected, both CURS1 mutants (G211F and G211W)
showed only weak activity in synthesizing curcuminoid fromferuloyl-CoA and cinnamoyldiketide-NAC (or 3-oxo-5-phe-nyl-4-pentenoic acid) (Fig. 4). Subsequently, the steady-statekinetics parameters of the G211F mutant were determined(Table 1). In the G211F mutant, the Km values for feruloyl-CoA were increased, and the kcat values were reduced mark-edly in both reactions. However, this change might have beencaused by the change in the structure of the catalytic triad. Toeliminate this possibility, we determined the crystal structureof CURS1 G211F at 2.5 Å resolution (Fig. 5). Except for thephenyl group generated by the mutation, the structure of theactive site pocket of CURS1 G211F was almost identical to
that of wild-type CURS1 and only slightly affected the orien-tation of Phe-215 (Fig. 5). As expected, the phenyl group gen-erated by the G211F mutation occupied the hydrophobic cav-ity around Phe-265 (Fig. 5). From these results, we concludedthat occupation of the cavity by the side chain of Phe-211generated by the G211F mutation resulted in a drastic de-crease in the curcuminoid synthesis activity of CURS1. Thisresult supported the idea that the hydrophobic cavity is re-sponsible for the hydrophobic interaction between CURS1and a �-keto acid, which is apparently essential for CURS1 touse an extender substrate lacking a CoA moiety. Thus, it wasvery likely that the reduced �-keto acid binding of the G211Fmutant resulted in the drastic decrease in the kcat values.However, we had no definite explanation for the increased Kmvalues of the G211F mutant for feruloyl-CoA. Conceivably,the G211F mutation inhibited feruloyl-CoA binding by re-stricting the flexibility of Phe-265. The flexibility of Phe-265was predicted to be important for starter substrate binding asdescribed below.Computational Modeling of CURS1 That Binds a Feruloyl
Moiety and a �-Keto Acid—To discuss the reaction catalyzedby CURS1, we used the molecular operating environmentprogram and constructed a model of CURS1 that contained aferuloyl moiety covalently attached to Cys-164 and bound5-(4-hydroxy-3-methoxy-phenyl)-3-oxo-4-pentenoic acid (a�-keto acid) in the active site pocket. The cavity that probablyaccommodates the starter substrate was found in the CURS1structure, but it was somewhat small to accommodate theferuloyl moiety. This observation suggested that a conforma-tional change of CURS1 could occur during starter substratebinding. Some type III PKSs have been predicted to requiresuch a conformational change during their reaction, and Phe-265 (in CHS) is predicted to be responsible for this change (4,
FIGURE 5. Orientation of the phenyl group generated by the G211F mu-tation. The white sticks indicate the side chains of the amino acid residuesaround the active site pocket of wild-type CURS1. Magenta sticks indicatethe phenyl group generated by the G211F mutation. Green sticks indicateside chains of the other amino acid residues of CURS1 G211F.
Decarboxylative Condensation of �-Keto Acid by CURS1
FEBRUARY 25, 2011 • VOLUME 286 • NUMBER 8 JOURNAL OF BIOLOGICAL CHEMISTRY 6665
by guest on March 6, 2020
http://ww
w.jbc.org/
Dow
nloaded from
6). The orientation of Phe-258 (which corresponds with Phe-265 in CHS) is also known to be different between chain Aand chain B in the crystal structure of BAS (19). These resultssuggested that flexibility of Phe-265 might be the commonfeature of type III PKSs. Thus, Phe-265 of CURS1 was as-sumed to move slightly to accommodate a feruloyl moiety(Fig. 6, A and B). The orientation of Phe-265 was predicted bythe Autodock program (25). We built several models in eachof which �-keto acid was differently located. However, wecould evaluate the significance of each model on the basis ofthe results of our biochemical analyses, which indicated thatCURS1 catalyzes decarboxylative condensation of �-keto acidusing the same mechanism as that for normal decarboxylativecondensation of malonyl-CoA by typical type III PKSs. Fi-nally, we selected a model as the most reliable one (Fig. 6B).The fact that the �-keto acid moiety in this model is locatedat a position similar to the malonic acid in the crystal struc-ture of CURS1 chain A (supplemental Fig. S6) also supportedthis model.In this model, the oxygen atom of the �-keto moiety was
located sufficiently close to the catalytic His-303 and Asn-336residues to interact with them (Fig. 6B). The �-carbon wasalso located close to the ketone carbon of the feruloyl moiety,so that the active anion formed by the decarboxylation of the�-keto acid could efficiently generate a carbon-carbon bondto the feruloyl moiety by nucleophilic attack (Fig. 6B). Thus,this model mimics the state immediately before decarboxyla-tive condensation. Importantly, in this model, the phenylgroup of the �-keto acid is located in the hydrophobic cavityaround Phe-265 and is surrounded by the side chains of Phe-215, Phe-265, and Phe-267 (Fig. 6B). As described above, thephenyl group of Phe-211 generated by the G211F mutationoccupied the cavity in the CURS1 G211F mutant that showedgreatly reduced curcuminoid synthesis activity. This resultsupported the present model in which the phenyl group of the�-keto acid interacts with the hydrophobic cavity.
DISCUSSION
In this study, we determined the crystal structure of CURS1followed by site-directed mutagenesis to examine the enzy-matic properties and a modeling study on substrate binding.
From these results, we conclude that CURS1 catalyzes head-to-head condensation of polyketide chains using the followingreactions (see Fig. 7). First, CURS1 catalyzes the transfer ofthe feruloyl moiety of feruloyl-CoA to the catalytic Cys-164.Diketide-CoA then enters the CoA-binding tunnel and is hy-drolyzed by an unknown mechanism. The resulting �-ketoacid subsequently accesses the catalytic triad and is used as anextender substrate. The hydrophobic cavity around Phe-265in the CoA-binding tunnel is responsible for the binding ofthe �-keto acid; the phenyl moiety of the �-keto acid interactswith the hydrophobic cavity. Finally, decarboxylative conden-sation of the �-keto acid with the feruloyl moiety results incurcumin formation. CURS1 catalyzes the decarboxylativecondensation of the �-keto acid using a mechanism identicalto that for normal decarboxylative condensation of malonyl-CoA in typical type III PKSs.We had assumed two possible mechanisms in which
CURS1 could use a �-keto acid as an extender substrate. First,a hydrogen bond network between a �-keto acid moiety andCURS1, which does not exist in other type III PKSs, mightallow the �-keto acid to be close to the active site withouthelp from the interaction between CoA and the CoA-bindingtunnel. Second, an additional hydrophobic pocket around thecatalytic triad might accommodate the phenyl group of the�-keto acid, so that the �-keto acid moiety could effectivelygain access to the catalytic center. The malonic acid that wascocrystallized in the active site pocket of the wild-type CURS1protein helped us to search for such a hydrogen bond networkto stabilize the �-keto moiety. However, we could not findany hydrogen bonds to interact with the malonic acid, exceptthose between the malonic acid and the catalytic His-303 andAsn-336 residues. Absence of a strong hydrogen bond net-work may explain the diversity of the orientation of malonicacid in the catalytic center of each CURS1 monomer. Theobservation that CURS1 could not use �-keto acids having ashort alkyl chain as an extender substrate also indicated thatsuch a hydrogen bond network should not exist between the�-keto acid moiety and CURS1. In contrast, we found aunique hydrophobic cavity in the CoA-binding tunnel andshowed that this cavity was responsible for the binding of the
FIGURE 6. Model for the binding of a feruloyl moiety and a �-keto acid in the active site pocket of CURS1. A, native structure of the active site pocketof CURS1. B, most reliable model structure. A feruloyl moiety and a �-keto acid are colored magenta and green, respectively.
Decarboxylative Condensation of �-Keto Acid by CURS1
6666 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 286 • NUMBER 8 • FEBRUARY 25, 2011
by guest on March 6, 2020
http://ww
w.jbc.org/
Dow
nloaded from
�-keto acid. We conclude that CURS1 obtained its ability touse an extender substrate that lacks the CoA moiety by pro-viding a hydrophobic cavity that accommodates a hydropho-bic moiety (a phenyl group or a long alkyl chain) of a �-ketoacid.In addition to the ability to use an extender substrate that
lacks a CoA moiety, CURS1 possesses two characteristic fea-tures. First, CURS1 catalyzes the hydrolysis of diketide-CoAs.From the kinetics analysis of the two curcuminoid synthesisreactions (one from feruloyl-CoA and cinnamoyldiketide-NAC and the other from feruloyl-CoA and 3-oxo-5-phenyl-4-pentenoic acid), the hydrolysis of diketide-CoA was revealedto be the rate-limiting reaction in the curcuminoid synthesisfrom feruloyl-CoA and the diketide-CoA. In BAS, diketide-CoA is predicted to be loaded on the catalytic Cys just as astarter substrate and then hydrolyzed by nucleophilic attackof a water molecule that was activated by the catalytic His(19). However, we assumed that the feruloyl moiety derivedfrom the starter substrate (feruloyl-CoA) would be bound tothe catalytic Cys of CURS1 when hydrolysis of a diketide-CoA(extender substrate) occurs (Fig. 7). Thus, CURS1 probablyuses a different catalytic mechanism to hydrolyze diketide-CoA. Second, CURS1 exhibited very weak activity for the nor-mal head-to-tail condensation of feruloyl-CoA with malonyl-CoA. Asn-306 in CURS1 may be responsible for its lowefficiency in the condensation of malonyl-CoA. The corre-sponding amino acid residue to Asn-306 is Gly or Ser in othertype III PKSs. Asn-306 is located at the wall of the CoA-bind-ing tunnel (Fig. 3A) and may inhibit the malonyl moiety ofmalonyl-CoA in approaching the catalytic center. In contrast,
a cavity that accommodates the feruloyl moiety is located onthe opposite side to the CoA-binding tunnel in the active sitepocket. Thus, Asn-306 may not affect starter substrate load-ing. Further experiments, including cocrystallization ofCURS1 with substrates and site-directed mutagenesis, arenecessary to determine the mechanisms of these two featuresof CURS1.Although the crystal structure of PqsD was previously re-
ported, the study was primarily focused on dihydroxyquino-line synthesis, which does not require �-keto acid condensa-tion, and its activity to condense a �-keto acid has not beenconfirmed to date (15). Thus, this study is the first report topredict the mechanism of condensation of a �-keto acid, anextender substrate that lacks the CoA moiety, by a ketosyn-thase. Because the head-to-head condensations of polyketidechains by ketosynthases exist not only in plants but also inbacteria and they seem to be preceded by the reaction similarto the CURS1 reaction, this study is of significance for under-standing the biochemistry of these natural products.
Acknowledgment—The synchrotron-radiation experiments wereperformed at the AR-NW12A beamline at the Photon Factory (Pro-posal 2008S2-001).
REFERENCES1. Austin, M. B., and Noel, J. P. (2003) Nat. Prod. Rep. 20, 79–1102. Abe, I., and Morita, H. (2010) Nat. Prod. Rep. 27, 809–8383. Ferrer, J. L., Austin, M. B., Stewart, C., Jr., and Noel., J. P. (2008) Plant
Physiol. Biochem. 46, 356–3704. Ferrer, J. L., Jez, J. M., Bowman, M. E., Dixon, R. A., and Noel, J. P.
FIGURE 7. Proposed mechanism of the reaction catalyzed by CURS1. First, the feruloyl moiety of feruloyl-CoA is transferred to the catalytic Cys of CURS1.Then a diketide-CoA interacts with CURS1 and is hydrolyzed to a �-keto acid. The resulting �-keto acid interacts with the catalytic center with the help ofthe hydrophobic cavity located around Phe-265 and is decarboxylated to form an active anion. The active anion attacks the carbon of ketone of the feruloylmoiety to form curcumin.
Decarboxylative Condensation of �-Keto Acid by CURS1
FEBRUARY 25, 2011 • VOLUME 286 • NUMBER 8 JOURNAL OF BIOLOGICAL CHEMISTRY 6667
by guest on March 6, 2020
http://ww
w.jbc.org/
Dow
nloaded from
(1999) Nat. Struct. Biol. 6, 775–7845. Jez, J. M., Austin, M. B., Ferrer, J., Bowman, M. E., Schroder, J., and
Noel, J. P. (2000) Chem. Biol. 7, 919–9306. Austin, M. B., Bowman, M. E., Ferrer, J. L., Schroder, J., and Noel, J. P.
(2004) Chem. Biol. 11, 1179–11947. Morita, H., Kondo, S., Oguro, S., Noguchi, H., Sugio, S., Abe, I., and
Kohno, T. (2007) Chem. Biol. 14, 359–3698. Katsuyama, Y., Matsuzawa, M., Funa, N., and Horinouchi, S. (2007)
J. Biol. Chem. 282, 37702–377099. Katsuyama, Y., Kita, T., Funa, N., and Horinouchi, S. (2009) J. Biol.
Chem. 284, 11160–1117010. Katsuyama, Y., Kita, T., and Horinouchi, S. (2009) FEBS Lett. 583,
2799–280311. Hertweck, C. (2009) Angew. Chem. Int. Ed. Engl. 48, 4688–471612. Ramirez-Ahumada, Mdel, C., Timmermann, B. N., and Gang, D. R.
(2006) Phytochemistry 67, 2017–202913. Kamo, T., Hirai, N., Tsuda, M., Fujioka, D., and Ohigashi, H. (2000) Bio-
sci. Biotechnol. Biochem. 64, 2089–209814. Brand, S., Holscher, D., Schierhorn, A., Svatos, A., Schroder, J., and
Schneider, B. (2006) Planta 224, 413–42815. Bera, A. K., Atanasova, V., Robinson, H., Eisenstein, E., Coleman, J. P.,
Pesci, E. C., and Parsons, J. F. (2009) Biochemistry 48, 8644–865516. Coleman, J. P., Hudson, L. L., McKnight, S. L., Farrow, J. M., 3rd, Calfee,
M. W., Lindsey, C. A., and Pesci, E. C. (2008) J. Bacteriol. 190,1247–1255
17. Zhang, Y. M., Frank, M. W., Zhu, K., Mayasundari, A., and Rock, C. O.(2008) J. Biol. Chem. 283, 28788–28794
18. Sukovich, D. J., Seffernick, J. L., Richman, J. E., Hunt, K. A., Gralnick,J. A., and Wackett, L. P. (2010) Appl. Environ. Microbiol. 76, 3842–3849
19. Morita, H., Shimokawa, Y., Tanio, M., Kato, R., Noguchi, H., Sugio,S., Kohno, T., and Abe, I. (2010) Proc. Natl. Acad. Sci. U.S.A. 107,669–673
20. Vagin, A., and Teplyakov, A. (1997) J. Appl. Crystallogr. 30, 1022–102521. Emsley, P., Lohkamp, B., Scott, W. G., and Cowtan, K. (2010) Acta Crys-
tallogr. D Biol. Crystallogr. 66, 486–50122. Murshudov, G. N., Vagin, A. A., and Dodson, E. J. (1997) Acta Crystal-
logr. D Biol. Crystallogr. 53, 240–25523. Jez, J. M., and Noel, J. P. (2000) J. Biol. Chem. 275, 39640–3964624. Jez, J. M., Ferrer, J. L., Bowman, M. E., Dixon, R. A., and Noel, J. P.
(2000) Biochemistry 39, 890–90225. Morris, G. M., Huey, R., and Olson, A. J. (2008)Current Protocols in Bioin-
formatics, chapt. 8, p. 14, unit 8, JohnWiley & Sons, Inc., Malden, MA
Decarboxylative Condensation of �-Keto Acid by CURS1
6668 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 286 • NUMBER 8 • FEBRUARY 25, 2011
by guest on March 6, 2020
http://ww
w.jbc.org/
Dow
nloaded from
HorinouchiYohei Katsuyama, Ken-ichi Miyazono, Masaru Tanokura, Yasuo Ohnishi and Sueharu
-Keto Acid by Curcumin SynthaseβCondensation of Structural and Biochemical Elucidation of Mechanism for Decarboxylative
doi: 10.1074/jbc.M110.196279 originally published online December 9, 20102011, 286:6659-6668.J. Biol. Chem.
10.1074/jbc.M110.196279Access the most updated version of this article at doi:
Alerts:
When a correction for this article is posted•
When this article is cited•
to choose from all of JBC's e-mail alertsClick here
Supplemental material:
http://www.jbc.org/content/suppl/2010/12/08/M110.196279.DC1
http://www.jbc.org/content/286/8/6659.full.html#ref-list-1
This article cites 24 references, 7 of which can be accessed free at
by guest on March 6, 2020
http://ww
w.jbc.org/
Dow
nloaded from
