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Biophysical investigation of type A PutAs reveals aconserved core oligomeric structureDavid A. Korasick1, Harkewal Singh2, Travis A. Pemberton2, Min Luo2, Richa Dhatwalia2 andJohn J. Tanner1,2
1 Department of Biochemistry, University of Missouri, Columbia, MO, USA
2 Department of Chemistry, University of Missouri, Columbia, MO, USA
Keywords
analytical ultracentrifugation; oligomerization;
proline catabolism; small-angle X-ray
scattering; X-ray crystallography
Correspondence
J. J. Tanner, Department of Biochemistry,
University of Missouri, Columbia, MO
65211, USA
Fax: +1 573 882 5635
Tel: +1 573 884 1280
E-mail: [email protected]
(Received 15 February 2017, revised 6 June
2017, accepted 12 July 2017)
doi:10.1111/febs.14165
Many enzymes form homooligomers, yet the functional significance of self-
association is seldom obvious. Herein, we examine the connection between
oligomerization and catalytic function for proline utilization A (PutA)
enzymes. PutAs are bifunctional enzymes that catalyze both reactions of
proline catabolism. Type A PutAs are the smallest members of the family,
possessing a minimal domain architecture consisting of N-terminal proline
dehydrogenase and C-terminal L-glutamate-c-semialdehyde dehydrogenase
modules. Type A PutAs form domain-swapped dimers, and in one case
(Bradyrhizobium japonicum PutA), two of the dimers assemble into a ring-
shaped tetramer. Whereas the dimer has a clear role in substrate channel-
ing, the functional significance of the tetramer is unknown. To address this
question, we performed structural studies of four-type A PutAs from two
clades of the PutA tree. The crystal structure of Bdellovibrio bacteriovorus
PutA covalently inactivated by N-propargylglycine revealed a fold and sub-
strate-channeling tunnel similar to other PutAs. Small-angle X-ray scatter-
ing (SAXS) and analytical ultracentrifugation indicated that Bdellovibrio
PutA is dimeric in solution, in contrast to the prediction from crystal pack-
ing of a stable tetrameric assembly. SAXS studies of two other type A
PutAs from separate clades also suggested that the dimer predominates in
solution. To assess whether the tetramer of B. japonicum PutA is necessary
for catalytic function, a hot spot disruption mutant that cleanly produces
dimeric protein was generated. The dimeric variant exhibited kinetic
parameters similar to the wild-type enzyme. These results implicate the
domain-swapped dimer as the core structural and functional unit of type A
PutAs.
Enzymes
Proline dehydrogenase (EC 1.5.5.2); L-glutamate-c-semialdehyde dehydrogenase (EC 1.2.1.88).
Databases
The atomic coordinates and structure factor amplitudes have been deposited in the Protein Data
Bank under accession number 5UR2. The SAXS data have been deposited in the SASBDB under
the following accession codes: SASDCP3 (BbPutA), SASDCQ3 (DvPutA 1.5 mg�mL�1),
SASDCX3 (DvPutA 3.0 mg�mL�1), SASDCY3 (DvPutA 4.5 mg�mL�1), SASDCR3 (LpPutA
3.0 mg�mL�1), SASDCV3 (LpPutA 5.0 mg�mL�1), SASDCW3 (LpPutA 8.0 mg�mL�1),
SASDCS3 (BjPutA 2.3 mg�mL�1), SASDCT3 (BjPutA 4.7 mg�mL�1), SASDCU3 (BjPutA
7.0 mg�mL�1), SASDCZ3 (R51E 2.3 mg�mL�1), SASDC24 (R51E 4.7 mg�mL�1), SASDC34
(R51E 7.0 mg�mL�1).
3029The FEBS Journal 284 (2017) 3029–3049 ª 2017 Federation of European Biochemical Societies
Introduction
The proline catabolic pathway in bacteria and eukary-
otes consists of the enzymes proline dehydrogenase
(PRODH) and L-glutamate-c-semialdehyde aldehyde
dehydrogenase (GSALDH) (Fig. 1) [1]. PRODH cat-
alyzes the FAD-dependent oxidation of L-proline to
D1-pyrroline-5-carboxylate (P5C). Hydrolysis of P5C
generates the substrate for GSALDH, L-glutamate-c-semialdehyde. GSALDH is an aldehyde dehydrogenase
(ALDH) superfamily enzyme that catalyzes the
NAD+-dependent oxidation of L-glutamate-c-semial-
dehyde to L-glutamate. In total, the pathway produces
a 4-electron oxidation. The electrons abstracted from
proline flow into the electron transport chain, while
the carbon skeleton of L-proline ultimately enters the
citric acid cycle as a-ketoglutarate. Proline catabolic
enzymes have been implicated in many aspects of
health and disease, including tumor suppression [2],
hyperprolinemia metabolic disorders [3], schizophrenia
susceptibility [4–8], life-span extension [9], production
of fungal virulence factors [10], and virulence and sur-
vival of pathogenic bacteria [11–14].PRODH and GSALSH (aka P5CDH and
ALDH4A1) are combined in some bacteria into a sin-
gle protein known as proline utilization A (PutA)
[1,15]. The name PutA refers to early studies of proline
utilization in bacteria, which led to the discovery that
a single gene encodes both PRODH and GSALDH
[16,17]. The covalent linking of two enzymes into one
polypeptide chain suggests the possibility of substrate
channeling, which can improve kinetic efficiency, pro-
tect reactive intermediates, and prevent crosstalk
between competing pathways, such as proline catabo-
lism and biosynthesis [18]. Indeed, kinetic evidence in
support of substrate channeling has been found for
several PutAs [19–23].PutAs can be classified according to global sequence
similarity and domain architectures (Fig. 2). The PutA
phylogenetic tree has three prominent branches (1, 2,
and 3), and there are three types of PutA domain
architectures (A, B, and C). Type A PutAs have N-
terminal PRODH and C-terminal GSALDH modules
(Fig. 2A). Type B PutAs have an additional C-term-
inal domain, which was recently shown to have the
ALDH superfamily fold [24,25]. Type C PutAs have
an N-terminal ribbon-helix-helix DNA-binding domain
in addition to the C-terminal domain. These PutAs
function as transcriptional repressors of the genes
encoding PutA and the proline transporter PutP.
Types A, B, and C PutAs are found in branch 1
(Fig. 2B). Only type A PutAs are found in branch 2,
and only type B PutAs are observed in branch 3.
Thus, combining the phylogenetic tree and domain
analysis yields five classes of PutA: 1A, 1B, 1C, 2A,
and 3B.
The PutAs studied to date show surprising diversity
in oligomeric state and quaternary structure. Crystal
structures and oligomeric states in solution have been
determined for the class 1A PutA from Bradyrhizo-
bium japonicum (BjPutA) [20], the class 2A PutA from
Geobacter sulfurreducens (GsPutA) [23], the class 1B
PutA from Sinorhizobium meliloti (SmPutA) [24], and
the class 3B PutA from Corynebacterium freiburgense
(CfPutA) [25]. These structures showed that the
PRODH and GSALDH active sites are spatially sepa-
rated and connected by a tunnel. Mutagenesis studies
showed the tunnel is used for substrate channeling [26].
Although the arrangement of domains within the pro-
tomer is similar in all three structures, the oligomeric
states and quaternary structures differ. GsPutA forms a
domain-swapped dimer in solution. In BjPutA – anothertype A PutA – two of the dimers assemble into a ring-
shaped tetramer. The type B PutAs SmPutA and
CfPutA form monomer–dimer equilibria in solution;
however, the quaternary structure of the dimer is com-
pletely different from the type A domain-swapped dimer
[24,25]. Small-angle X-ray scattering (SAXS) showed
that the class 1C PutA from Escherichia coli forms yet a
third type of dimer, which is mediated by the DNA-
binding domain [27]. The functional relevance of these
various oligomeric states and quaternary structures has
not been systematically studied in detail.
The observation of different oligomeric states in
two-type A PutAs from different branches of the phy-
logenetic tree motivated us to study the self-association
of other type A PutAs to understand the relationship
between oligomeric state and catalytic function. In
particular, we sought to determine whether oligomeric
state distinguishes class 1A from class 2A and whether
Abbreviations
ALDH, aldehyde dehydrogenase; BbPutA, Bdellovibrio bacteriovorus proline utilization A; BCA, bicinchoninic acid assay; BjPutA,
Bradyrhizobium japonicum proline utilization A; CfPutA, Corynebacterium freiburgense proline utilization A; DvPutA, Desulfovibrio vulgaris
proline utilization A; GSALDH, L-glutamate-c-semialdehyde dehydrogenase; GsPutA, Geobacter sulfurreducens proline utilization A; LpPutA,
Legionella pneumophila proline utilization A; P5C, D1-pyrroline-5-carboxylate; PDB, Protein Data Bank; PRODH, proline dehydrogenase; PutA,
proline utilization A; SAXS, small-angle X-ray scattering; SEC, size exclusion chromatography; SmPutA, Sinorhizobiummeliloti proline
utilization A; TEVP, tobacco etch virus protease; THP, tris(3-hydroxypropyl)phosphine.
3030 The FEBS Journal 284 (2017) 3029–3049 ª 2017 Federation of European Biochemical Societies
Conservation of a core dimer in type A PutAs D. A. Korasick et al.
tetramerization of BjPutA is required for catalytic
activity.
Herein, we report a crystal structure of the class 2A
PutA from Bdellovibrio bacteriovorus (BbPutA), along
with SAXS data for BbPutA and three other type A
PutAs (from classes 1A and 2A). We also employed
analytical ultracentrifugation, size-exclusion chro-
matography, and enzyme kinetics for an extensive bio-
physical characterization of these enzymes. The data
suggest type A PutAs are predominantly dimeric in
solution, and global sequence identity is not a reliable
indicator of the oligomeric state. Further, we investi-
gate the necessity of higher order oligomeric states by
generating a dimeric hot spot mutant of the tetrameric
BjPutA. Overall, these results reveal a conserved dimer
as the essential oligomer required for catalysis and
channeling in type A PutAs. Our results also demon-
strate the challenges of predicting the solution oligo-
meric state from crystal packing.
Results
Crystal structure of covalently inactivated
BbPutA
The crystal structure of BbPutA inactivated by the
mechanism-based inactivator N-propargylglycine
(NPPG) was determined at 2.2 �A resolution (Table 1).
Similar to other PutAs, BbPutA has spatially sepa-
rated PRODH and GSALDH active sites (Fig. 3A).
The PRODH active site resides at the C termini of the
strands of a (ba)8 barrel. The GSALDH active site is
located in the crevice between the Rossmann dinu-
cleotide–binding domain and the GSALDH catalytic
domain. In addition to the catalytic domains, the fold
includes an N-terminal arm domain and an a-domain
in the PRODH half of the chain, as well as an
oligomerization flap at the C terminus. These ancillary
domains are also observed in other type A PutA struc-
tures. The root mean square deviation between
BbPutA and GsPutA is 1.1 �A, as expected for two
proteins with high sequence identity (47%, Table 2).
Fig. 1. The reactions catalyzed by PutA. PutAs consist of two modules that catalyze the oxidation of L-proline to L-glutamate. The first
module, proline dehydrogenase (PRODH), utilizes FAD to catalyze the conversion of L-proline to D1-pyrroline-5-carboxylate, which is subject
to nonenzymatic hydrolysis to L-glutamate-c-semialdehyde. The second module, L-glutamate-c-semialdehyde dehydrogenase (GSALDH),
performs the NAD+-dependent oxidation of L-glutamate-c-semialdehyde to the final product, L-glutamate.
Fig. 2. Classification of PutAs according to domain architecture
and global sequence identity. (A) The three domain architectures of
PutAs. RHH, ribbon-helix-helix; ALDHSF, aldehyde dehydrogenase
superfamily. The small N-terminal domain of type C PutAs is a
ribbon–helix–helix DNA-binding domain. (B) Phylogenetic tree
based on a global sequence alignment of PutAs. Architecture types
A, B, and C are indicated by black, blue, and red font, respectively.
The PutAs mentioned in the text are noted in large font. The
alignment was calculated with CLUSTAL OMEGA [75] and visualized
with DRAWTREE [76]. Abbreviation not listed in the text: EcPutA,
Escherichia coli proline utilization A.
3031The FEBS Journal 284 (2017) 3029–3049 ª 2017 Federation of European Biochemical Societies
D. A. Korasick et al. Conservation of a core dimer in type A PutAs
The deviations of BbPutA from BjPutA (class 1A) and
type B PutAs are also low (1.8–2.0 �A), despite sharing
only ~ 30% sequence identity with these proteins. This
result attests to the high structural conservation of the
PutA fold.
BbPutA has a substrate-channeling tunnel. The
FAD in the PRODH active site and the catalytic Cys
(Cys778) of the GSALDH site are separated by a lin-
ear distance of 45 �A and connected by a 65-�A curved
tunnel that varies in diameter from 1.5 �A near the
active sites to 4.5 �A in the middle section (Fig. 3A).
The dimensions of the tunnel are similar to those of
other PutAs. A coupled PRODH-GSALDH enzyme
activity assay, which tests both active sites and pro-
vides preliminary evidence of substrate channeling [26],
Table 1. Data collection and refinement statisticsa.
Space group P21221
Beamline APS 24-ID-E
Unit cell parameters (�A) a = 144.3, b = 158.6, c = 221.1
Wavelength (�A) 0.97920
Resolution (�A) 128.9–2.23 (2.35–2.23)
Observations 930 561
Unique reflections 241 211
Rmerge (I) 0.095 (0.493)
Rmeas (I) 0.124 (0.644)
Rpim (I) 0.061 (0.320)
Mean I/r 10.0 (2.4)
Completeness (%) 97.9 (98.3)
Multiplicity 3.9 (3.9)
No. of protein residues 3829
No. of atoms
Protein 29 525
FAD 212
Modified Lys 48
Water 1048
Rcryst 0.183 (0.230)
Rfreeb 0.228 (0.291)
rmsd bond lengths (�A) 0.008
rmsd bond angles (°) 0.872
Ramachandran plotc
Favored (%) 97.95
Outliers (%) 0.00
Clashscore (PR)c 1.73 (100)
MolProbity score (PR)c 0.94 (100)
Average B (�A2)
Protein 31.2
FAD 31.4
Modified Lys 43.0
Water 30.2
Coordinate error (�A)d 0.27
PDB code 5UR2
a Values for the outer resolution shell of data are given in parenthe-
ses. b 5% test set. c From MOLPROBITY. The percentile ranks (PR) for
Clashscore and MolProbity score are given in parentheses. d Maxi-
mum likelihood-based coordinate error estimate from PHENIX.
A
B
C E
D
Fig. 3. Structure of BbPutA. (A) A protomer of BbPutA with the
domains colored according to the domain diagram. The pink surface
represents the substrate-channeling tunnel, which connects the two
active sites. The FAD is shown in yellow sticks. Catalytic Cys778 is
drawn in spheres. (B) The domain-swapped dimer of BbPutA. The
two protomers have different colors. The molecular two-fold axis is
vertical. (C) Close-up view of a portion of the dimer interface where
the oligomerization flap of one protomer covers the substrate-
channeling tunnel of the opposite protomer. (D) Surface
representation of the dimer interface shown in panel C, highlighting
how dimerization seals the substrate-channeling tunnel from the
bulk medium. (E) Results of a PRODH-GSALDH-coupled assay for
BbPutA. The reaction mixture contained BbPutA (0.2 lM), proline
(40 mM), menadione bisulfite (0.1 mM), and NAD+ (0.2 mM) in a
buffer containing 50 mM potassium phosphate, 25 mM NaCl, and
10 mM MgCl2 at pH 7.5. Production of NADH was monitored at
340 nm. The data points represent the average of assays performed
in triplicate. [Colour figure can be viewed at wileyonlinelibrary.com]
3032 The FEBS Journal 284 (2017) 3029–3049 ª 2017 Federation of European Biochemical Societies
Conservation of a core dimer in type A PutAs D. A. Korasick et al.
confirmed that both actives sites are functional
(Fig. 3E). In addition, no lag phase is apparent in the
time-dependence of NADH production, consistent
with the presence of a functional substrate-channeling
tunnel.
The structure of BbPutA was determined from a
crystal of the enzyme that had been inactivated with
NPPG (Fig. 4A, 1). Previous studies of NPPG-inacti-
vated PRODHs and PutAs show that inactivation is
due to a covalent link between the FAD N5 atom and
a conserved active site Lys (Fig. 4A, 2). Electron den-
sity maps for BbPutA show evidence of this inactiva-
tion mechanism. An electron density feature connects
the e-amino group of Lys196 with the N5 atom of the
FAD (Fig. 4B). The strength of this feature varied
among the four chains in the asymmetric unit, with
the strongest density observed in chain D (Fig. 4). The
electron density feature could be modeled satisfactorily
as a 3-carbon link between Lys196 and the N5 of the
FAD consistent with the presence of a covalent NPPG
modification in the active site.
The FAD exhibits the structural hallmarks of reduc-
tion. Previous studies revealed that NPPG locks the
flavin into a conformation that resembles the 2-elec-
tron reduced state [28]. These features are seen in the
BbPutA structure. In particular, the isoalloxazine moi-
ety exhibits a butterfly bend of 25° (si face convex)
(Fig. 4B). For reference, the bend angles of other
NPPG-inactivated PutAs and PRODHs are 25°–35°[23,28,29]. Furthermore, the ribityl chain conformation
of NPPG-inactivated BbPutA is indicative of reduced
PutA/PRODH. The 20-OH and 30-OH groups of the
inactivated FAD are rotated toward the pyrimidine
side of the isoalloxazine, while the 40-OH is beneath
the dimethylbenzene ring (Fig. 4C). This conformation
is also observed in other NPPG-inactivated or dithion-
ite-reduced PutAs [23,28] and PRODHs [29,30].
Solution oligomeric state analysis of BbPutA by
SAXS and analytical ultracentrifugation
The oligomeric state and quaternary structure of
BbPutA in solution were determined using SAXS and
analytical ultracentrifugation (Fig. 5, Table 3). Guinier
analysis of experimental SAXS data yields a radius of
gyration (Rg) of 45 �A (Fig. 5A). For reference, the 2-
body assembly in the crystallographic asymmetric unit
also has Rg of 45 �A (Fig. 3B). The real space Rg from
calculations of the distance distribution function is
45.9–46.7 �A for assumed maximum particle dimension
(Dmax) of 140–150 �A (Fig. 5B, Table 3). Thus, the
reciprocal space and real space radii of gyration are in
good agreement. The oligomeric state was estimated
using the volume of correlation method [31]. This
analysis yields molecular mass (Mr) of 190 kDa, which
is within 13% of the theoretical Mr of a dimer
(218 kDa, Table 3). Similarly, Mr estimated from the
SAXS MoW2 server [32] is 226 kDa, which is within
4% of the dimer (Table 3). Altogether, the SAXS data
are consistent with BbPutA forming a dimer under the
solution conditions and protein concentration
(1.8 mg�mL�1) used for SAXS.
The BbPutA crystal lattice was inspected using PDBe-
PISA [33] to identify plausible oligomers. This analysis
revealed two stable assemblies, including the classic
type A PutA domain-swapped dimer (Rg = 45 �A,
Figs 3B and 5C) and a ring-shaped tetramer
(Rg = 54 �A, Fig. 5D). The predicted tetramer is gener-
ated by a crystallographic 2-fold rotation applied to
the domain-swapped dimer. Thus, the tetramer is a
Table 2. Pairwise amino acid sequence identities of type A PutAs.
LpPutA BjPutA BbPutA GsPutA DvPutA
LpPutA 100 50 31 31 30
BjPutA 100 32 31 29
BbPutA 100 47 48
GsPutA 100 67
Fig. 4. Covalent modification of the FAD. (A) Structures of (1)
NPPG and (2) the covalently modified FAD resulting from
inactivation by NPPG. (B) Electron density evidence of inactivation
of BbPutA by NPPG. This view of the N5 edge of the isoalloxazine
shows the 25° butterfly bend induced by inactivation. The mesh
represents a simulated annealing Fo-Fc omit map contoured at 2.5
r. (C) Electron density for the modified FAD of BbPutA. The mesh
represents a simulated annealing Fo-Fc omit map contoured at
2.5 r.
3033The FEBS Journal 284 (2017) 3029–3049 ª 2017 Federation of European Biochemical Societies
D. A. Korasick et al. Conservation of a core dimer in type A PutAs
dimer-of-dimers and has 222 point group symmetry.
We note this tetramer resembles the one formed by
BjPutA in solution; however, the BbPutA tetramer is
slightly larger (Rg of 54 �A vs. 51 �A for BjPutA). In
addition, PDBePISA analysis returned an octamer
consisting of two of the 54 �A tetramers, which was
classified in the uncertain region of complex formation
criteria (Rg = 68 �A). The experimental Rg of 45–47 �A
suggests that the dimer is the predominant species in
solution. FoXS [34,35] was used to assess the agree-
ments of the dimer and tetramer models to the
experimental curve. The scattering curve calculated
from the dimer has a goodness-of-fit parameter (v) of1.3, whereas the curve calculated from the crystallo-
graphic tetramer has a much larger v of 18 (Fig. 5A).
Monomer–dimer and dimer–tetramer equilibria were
explored using MultiFoXS [35]. MultiFoXS returned
neither monomer–dimer nor dimer–tetramer 2-body
fits, indicating that 100% dimer provides the best
interpretation of the SAXS data. Finally, shape recon-
struction with DAMMIF [36] produced a shape consistent
with the crystallographic dimer (Fig. 5C). In summary,
Fig. 5. SAXS and analytical ultracentrifugation of BbPutA. (A) Experimental SAXS curve measured at 1.8 mg�mL�1 (open circles). The inset
shows a Guinier plot. Theoretical curves calculated from the BbPutA dimer (C) and tetramer (D) are shown in solid red (FoXS v = 1.3) and
red dashes (FoXS v = 18.1), respectively. (B) Experimental SAXS distance distribution function. (C) The crystallographic dimer of BbPutA.
The surface represents the shape reconstruction from DAMMIF. The correlation coefficient between the crystal structure and the shape
reconstruction volumetric map is 0.85. (D) The BbPutA tetramer predicted by PDBePISA from analysis of crystal packing. (E) Sedimentation
velocity analysis for BbPutA at 4 mg�mL�1 (~ 37 lM).
3034 The FEBS Journal 284 (2017) 3029–3049 ª 2017 Federation of European Biochemical Societies
Conservation of a core dimer in type A PutAs D. A. Korasick et al.
SAXS suggests that BbPutA is primarily dimeric in
solution, and furthermore, the 2-body assembly in the
asymmetric unit is the predominant species formed in
solution.
Analytical ultracentrifugation was employed to
determine whether BbPutA self-associates into higher
oligomeric states at a higher concentration than was
used in SAXS. A sedimentation velocity experiment
performed at 4 mg�mL�1 (~ 37 lM) revealed a distri-
bution of sedimentation coefficients with a major peak
at approximately 6.4 S. This apparent sedimentation
coefficient corresponds to Mr of approximately
225 kDa (Fig. 5E). The expected Mr of a BbPutA
dimer is 218.6 kDa.
Analytical ultracentrifugation was also used to test
whether inactivation with NPPG induces tetrameriza-
tion. This experiment was motivated by the observa-
tion of an apparent tetramer in the crystal structure of
NPPG-inactivated BbPutA (Fig. 5D). Sedimentation
velocity analysis of NPPG-inactivated BbPutA
revealed one major peak corresponding to Mr of
225 kDa (Fig. 5E), showing that inactivation does not
promote tetramerization in solution.
Finally, sedimentation velocity was performed on
BbPutA in the presence of the active site ligands L-te-
trahydro-2-furoic acid (THFA, 10 mM) and NAD+
(1 mM). THFA is a competitive inhibitor (competitive
with proline) of PutAs and is known to occupy the
proline-binding site of PutAs and monofunctional
PRODHs [23,24,30,37,38]. NAD+ is the cofactor of
the GSALDH reaction. This experiment was moti-
vated by our recent observation that THFA and
NAD+ induce monomers of the class 3B CfPutA to
form dimers [25]. The c(s) distribution obtained for
BbPutA under this condition exhibited a single c(s)
peak at 6.4 S, corresponding to Mr of 225 kDa
Table 3. Structural and molecular mass (Mr) parameters from SAXS for BbPutA, DvPutA and LpPutA.
BbPutA 1.8 mg�mL�1
Rg from Guinier (�A) 44.9 � 0.4
I(0) from Guinier (�A�1) 1130 � 7
Dmax (�A) 140–150
Rg from P(r) (�A)a 45.9–46.7
I(0) from P(r) (�A�1)a 1117–1132
Porod volume (�A3)a 288 000–289 000
Volume of correlation, Vc (�A2)b 1034
Mr from Vc (kDa)b 190
Mr from MoW2 (kDa)c 226
Monomeric Mr from sequence 109
DvPutA 1.5 mg�mL�1 3.0 mg�mL�1 4.5 mg�mL�1
Rg from Guinier (�A) 43.6 � 0.4 43.6 � 0.3 43.9 � 0.3
I(0) from Guinier (�A�1) 681 � 4 1600 � 8 2319 � 11
Dmax (�A) 150–160 150–160 150–160
Rg from P(r) (�A)a 46.1–47.0 45.3–45.7 45.7–46.5
I(0) from P(r) (�A�1)a 699–703 1623–1627 2350–2364
Porod volume (�A3)a 292 000–294 000 294 000–295 000 294 000–295 000
Volume of correlation, Vc (�A2)b 994 1020 1010
Mr from Vc (kDa)b 180 190 180
Mr from MoW2 (kDa)c 213 224 222
Monomeric Mr from sequence 112 112 112
LpPutA 3 mg�mL�1 5 mg�mL�1 8 mg�mL�1
Rg from Guinier (�A) 45.5 � 0.2 45.9 � 0.1 46.3 � 0.1
I(0) from Guinier (�A�1) 471 � 1 1010 � 1 1475 � 0.2
Dmax (�A) 150–160 150–166 150–170
Rg from P(r) (�A)a 46.5–46.9 46.6–46.9 46.7–47.2
I(0) from P(r) (�A�1)a 473–475 1014–1018 1470–1481
Porod volume (�A3)a 290 000–291 000 297 000–298 000 294 000–296 000
Volume of correlation, Vc (�A2)b 1013 1035 1030
Mr from Vc (kDa)b 180 190 190
Mr from MoW2 (kDa)c 230 241 231
Monomeric Mr from sequence 116 116 116
a From calculations of P(r) using PRIMUS [64] with the indicated range of Dmax.b Calculated with Scatter 3.0 [70]. c Calculated from the
method of Fischer et al. [32].
3035The FEBS Journal 284 (2017) 3029–3049 ª 2017 Federation of European Biochemical Societies
D. A. Korasick et al. Conservation of a core dimer in type A PutAs
(Fig. 5E), showing that the binding of THFA and
NAD+ does not induce higher order oligomerization.
Taken together, the SAXS and analytical ultracentrifu-
gation results are consistent with BbPutA being pri-
marily dimeric under solution conditions that are
relevant to catalytic function.
SAXS analysis of two other type A PutAs
To further explore the oligomerization of type A
PutAs, SAXS was performed on the class 2A PutA
from Desulfovibrio vulgaris (DvPutA) and the class 1A
PutA from Legionella pneumophila (LpPutA). DvPutA
is 48% identical in amino acid sequence to BbPutA
and 67% identical to GsPutA (Table 2). Such high
sequence homology is expected from PutAs of the
same clade (branch 2, Fig. 2B). In contrast, LpPutA, a
branch 1 PutA, is only 30% identical to the class 2A
PutAs, but 50% identical to the class 1A BjPutA
(Table 2). Analysis of DvPutA and LpPutA provides
two more data points on the relationship between
sequence and oligomeric state for type A PutAs.
SAXS curves measured at three different concentra-
tions of DvPutA and LpPutA are shown in Fig. 6.
SAXS-derived parameters are listed in Table 3. The
average Rg from Guinier analysis is 44 �A for DvPutA
and 46 �A for LpPutA. These values agree more closely
with the Rg of 45 �A of dimeric BbPutA than with the
Rg expected for a tetrameric type A PutA (51–54 �A).
The distance distribution functions of DvPutA and
LpPutA resemble that of BbPutA. For all three pro-
teins, the distribution has a major peak near the real-
space vector length (r) of 40 �A followed by a shoulder
peak at r = 80–100 �A (Figs 5B and 6B,D), suggesting
the three proteins share a common quaternary struc-
ture. Further, the SAXS-derived molecular masses of
DvPutA and LpPutA based on the volume of correla-
tion are within 20% of the theoretical dimer masses
for each protein (Table 3). Moreover, the masses from
the SAXS MoW2 server are within 4% of the theoreti-
cal dimer mass (Table 3). Note also the Porod volumes
for DvPutA and LpPutA (290 000–298 000 �A3) are
similar to that of BbPutA (289 000 �A3) and almost
two times smaller than expected for tetrameric PutA
(~ 560 000 �A3). The theoretical SAXS curves calcu-
lated from domain-swapped dimeric homology models
show good agreement with the experimental profiles in
the region of q = 0–0.1 �A�1 (Fig. 6). We note that this
region is critical for distinguishing between dimeric
and tetrameric PutAs (see Fig. 5A and references
[20,23]). Monomer–dimer and dimer–tetramer equilib-
ria were explored using MultiFoXS [35]. As with
BbPutA, MultiFoXS returned neither monomer–dimer
nor dimer–tetramer 2-body fits, indicating that 100%
dimer provides the best interpretation of the SAXS
data for DvPutA and LpPutA. Finally, shape recon-
structions generated shapes that are consistent with
the dimeric models and do not resemble a ring-shaped
particle (Fig 6A,C). Altogether, the SAXS results sug-
gest that DvPutA and LpPutA are dimeric in solution
and form the classic type A PutA domain-swapped
dimer.
Identification of the core functional oligomer of
BjPutA
All type A PutAs studied thus far appear to be dimeric
in solution except for BjPutA, which is tetrameric
(Fig. 7A) [20]. To better understand whether the tetra-
meric assembly is necessary for BjPutA function, we
attempted to generate a hot spot mutant to disrupt
tetramerization. Structural analysis of BjPutA revealed
Arg51 as potentially important for stabilizing the tetra-
mer. Arg51 is located in the dimer–dimer interface on
a flexible loop that connects the N-terminal arm to the
a-domain (Fig. 7B). Although Arg51 has weak elec-
tron density and appears to make no strong interdo-
main hydrogen bonds or ion pairs, its location in the
center of the dimer–dimer interface nevertheless sug-
gested that it could be important for tetramer forma-
tion. Analysis with PDBePISA indicates that Arg51
contributes 76 �A2 of surface area to the tetramer inter-
face, which makes it the second-largest contributor,
behind only Tyr474 (99 �A2). Interestingly, Arg does
not appear at this position in the sequences of the
dimeric type A PutAs studied so far; rather, it is
replaced with Glu in LpPutA, Gln in BbPutA, and
Gly in GsPutA and DvPutA. Therefore, we generated
a mutant, R51E, to reflect the charge reversal seen in
LpPutA. We note the introduction of Glu51 in BjPutA
results in three consecutive acidic residues: Glu51-
Asp52-Asp53.
To understand any effects of this mutation on the
quaternary structure of BjPutA, we first purified wild-
type BjPutA and subjected it to analysis by sedimenta-
tion velocity. Initial sedimentation velocity studies of
wild-type BjPutA revealed a major peak near apparent
sedimentation coefficient of 10.8 S (Fig. 8A), which
corresponds to Mr of 429 kDa (Fig. 8B). The pre-
dicted Mr of the BjPutA tetramer is also 429 kDa.
Wild-type BjPutA was also studied with SAXS. The
SAXS curves for wild-type BjPutA show pronounced
trough and peak features in the region of q = 0.05–0.1 �A�1 (Fig. 8C). We have shown previously that
these features are diagnostic of the ring-shaped tetra-
meric form of type A PutA [20,23]. Note these
3036 The FEBS Journal 284 (2017) 3029–3049 ª 2017 Federation of European Biochemical Societies
Conservation of a core dimer in type A PutAs D. A. Korasick et al.
diagnostic features are absent in the SAXS curves for
all the other PutAs studied here. Also, the distance dis-
tribution function of wild-type BjPutA is very different
from those of the other proteins in this study
(Fig. 8D). The distribution function of wild-type
BjPutA indicates a most-probable real-space vector
length of r ~ 80 �A (Fig. 8D), compared to approxi-
mately 40 �A for the other PutAs (Figs 5B and 6B,D).
The SAXS Rg of 51–53 �A for wild-type BjPutA
(Table 4) is in good agreement with the Rg of 51 �A
calculated from the BjPutA tetramer. The molecular
masses derived from SAXS are within 0.5–11% of the
expected mass of a tetramer (Table 4). The SAXS
curve calculated from the BjPutA tetramer shows good
agreement with the experimental curves (v = 4.3–9.1,Fig. 8C). The fits could be improved somewhat (to
v = 1.6–4.4) by using a tetramer : dimer ensemble in
MultiFoXS (Fig. 8C). The optimal ratio of
Fig. 6. SAXS analysis of DvPutA and LpPutA. (A) Experimental SAXS curves for DvPutA (open circles) at three concentrations: 1.5, 3.0, and
4.5 mg�mL�1. The inset shows Guinier plots. The red curve was calculated from a homology model of the DvPutA domain-swapped dimer
(shown in the inset). The v values obtained from FoXS fits of the theoretical scattering curve of the homology model to the experimental
scattering data are as follows: v = 1.3 (1.5 mg�mL�1), v = 1.7 (3.0 mg�mL�1), and v = 2.3 (4.5 mg�mL�1). A homology model of the DvPutA
dimer is shown inside the DAMMIF shape reconstruction. The correlation coefficient between the homology model and the shape
reconstruction volumetric map is 0.79. (B) Experimental SAXS distance distribution functions for DvPutA. (C) SAXS curves for LpPutA (3, 5,
8 mg�mL�1). The inset shows Guinier plots. The red curve was calculated from a homology model of the LpPutA domain-swapped dimer
(shown in the inset). The v values obtained from FoXS fits of the theoretical scattering curve of the homology model to the experimental
scattering data are as follows: v = 3.3 (3.0 mg�mL�1), v = 4.9 (5.0 mg�mL�1), and v = 8.7 (8.0 mg�mL�1). A homology model of the LpPutA
dimer is shown inside the DAMMIF shape reconstruction. The correlation coefficient between the homology model and the shape
reconstruction volumetric map is 0.78. (D) Experimental SAXS distance distribution functions for LpPutA.
3037The FEBS Journal 284 (2017) 3029–3049 ª 2017 Federation of European Biochemical Societies
D. A. Korasick et al. Conservation of a core dimer in type A PutAs
tetramer : dimer ranged from 74%:26% for the lowest
concentration sample to 81%:19% for the highest con-
centration sample. Thus, the fitting calculations are
consistent with BjPutA being predominantly tetrameric
in solution. Finally, shape reconstruction calculations
using the SAXS data for the highest concentration
sample returned a ring-shaped particle consistent with
the crystallographic tetramer of BjPutA (Fig. 8C). In
summary, the SAXS data show that wild-type BjPutA
exists in solution primarily as a ring-shaped tetramer,
in agreement with our previously crystallographic,
SAXS, and centrifugation studies [20,23].
The mutation of Arg51 to Glu profoundly changes
the oligomeric structure of BjPutA. To determine the
effects of the R51E mutation on the oligomeric state
of BjPutA, we subjected the BjPutA R51E mutant
variant to the same analysis as wild-type BjPutA. Sedi-
mentation velocity experiments on BjPutA R51E
revealed a major peak at apparent sedimentation coef-
ficient of 6.5 S, which corresponds to Mr of 216 kDa
(Fig 8A,B). The theoretical Mr for the BjPutA dimer
is 214.7 kDa, which is within 0.6% of the result from
sedimentation velocity, suggesting BjPutA R51E is
dimeric in solution. SAXS analysis of BjPutA R51E at
three protein concentrations revealed experimental
curves statistically very similar (v = 0.9–2.1) to the
theoretical curves generated by FoXS using the
crystallographic domain-swapped dimer (Fig. 8E).
Monomer–dimer and dimer–tetramer equilibria were
explored using MultiFoXS [35]. MultiFoXS returned
neither monomer–dimer nor dimer–tetramer 2-body
fits, indicating that 100% dimer provides the best
interpretation of the SAXS data for R51E. Guinier
analysis returned Rg of 45 �A, consistent with the Rg of
dimeric type A PutAs (Table 4). The real-space Rg of
45–46 �A agrees well with the Guinier Rg. The P(r) dis-
tribution for R51E (Fig. 8F) is distinctly different
from that of wild-type BjPutA (Fig. 8D). Further, fit-
ting a representative experimental R51E SAXS curve
to the BjPutA crystallographic dimer yields a greatly
improved statistical fit (v = 1.3) compared with fitting
the experimental data to the BjPutA tetramer (v = 51;
Fig. 9A). Moreover, the experimental P(r) distribu-
tions of wild-type BjPutA and R51E are qualitatively
very different (Fig. 9B). In fact, the area under the dis-
tance distribution function of R51E is approximately
Fig. 7. Structural context of Arg51 of BjPutA. (A) The BjPutA
tetramer with the four protomers in different colors. The domain-
swapped dimers are colored red-cyan and gold-blue. Arg51 is
shown in spheres in the right-hand image. The boxed region is
expanded in panel B. (B) Close-up view of the dimer–dimer
interface. The dashed curves represent disordered residues 52–53.
Fig. 8. Solution oligomeric state analysis of BjPutA and BjPutA R51E. (A) The distribution of apparent sedimentation coefficients from
sedimentation velocity observed for BjPutA (28 lM, black) or BjPutA R51E (28 lM, red). (B) The distribution of molecular masses from
sedimentation velocity observed for BjPutA (28 lM, black) or BjPutA R51E (28 lM, red). (C) SAXS curves measured at three wild-type BjPutA
protein concentrations (2.3, 4.7, 7.0 mg�mL�1). The inset shows Guinier plots. The theoretical SAXS curve calculated from the crystallographic
tetramer (inset) is shown in cyan [v values of 4.3 (2.3 mg�mL�1), 7.3 (4.7 mg�mL�1), and 9.1 (7.0 mg�mL�1)]. The theoretical curves obtained
from MultiFoXS assuming a mixture of the crystallographic tetramer and dimer are shown in red [v values of 1.6 (2.3 mg�mL�1), 3.3
(4.7 mg�mL�1), and 4.4 (7.0 mg�mL�1)]. The optimal tetramer : dimer compositions from MultiFoXS are 74%:26% (2.3 mg�mL�1), 77%:23%
(4.7 mg�mL�1), and 81%:19% (7.0 mg�mL�1). The crystallographic tetramer of BjPutA is shown inside the DAMMIF shape reconstruction. The
correlation coefficient between the tetramer and the shape reconstruction volumetric map is 0.78. (D) Experimental SAXS distance distribution
functions for wild-type BjPutA. (E) SAXS curves measured at three BjPutA R51E protein concentrations. The inset shows Guinier plots. The
theoretical SAXS curve calculated from the BjPutA domain-swapped dimer (inset) is shown in red. The v values obtained from FoXS fits of the
theoretical scattering curve of the crystallographic dimer to the experimental scattering data are as follows: v = 0.85 (2.3 mg�mL�1), v = 1.3
(4.7 mg�mL�1), and v = 2.5 (7.0 mg�mL�1). The crystallographic dimer of BjPutA is shown inside the DAMMIF shape reconstruction. The
correlation coefficient between the tetramer and the shape reconstruction volumetric map is 0.83. (F) Experimental SAXS distance distribution
functions for BjPutA R51E.
3038 The FEBS Journal 284 (2017) 3029–3049 ª 2017 Federation of European Biochemical Societies
Conservation of a core dimer in type A PutAs D. A. Korasick et al.
54% of the area under the distance distribution func-
tion of wild-type BjPutA at the same protein concen-
tration, consistent with R51E being half the size of
wild-type BjPutA (Fig. 9B). Overall, these results sug-
gest that the R51E mutation cleanly disrupts tetramer-
ization, resulting in consistently dimeric BjPutA.
3039The FEBS Journal 284 (2017) 3029–3049 ª 2017 Federation of European Biochemical Societies
D. A. Korasick et al. Conservation of a core dimer in type A PutAs
To determine whether the R51E mutation has an
effect on the catalytic activity, we performed the cou-
pled PRODH-GSALDH activity assay, which reports
on both catalytic activities and provides information
on substrate channeling. Previous results have shown
that at high concentrations of proline, kinetic data for
the coupled PRODH-GSALDH assay fit to a substrate
inhibition model [26]. Substrate inhibition was also
observed here for both wild-type BjPutA and R51E
(Fig. 10A,B). Using proline as the variable substrate,
wild-type BjPutA displayed a Km of 4 mM, kcat of
0.31 s�1, and inhibition constant for proline (Ki) of
400 mM (Fig. 10A, Table 5). Similarly, the kinetic
parameters for R51E are Km = 10 mM, kcat = 0.4 s�1,
and Ki of 220 mM (Fig. 10B, Table 5). The catalytic
efficiencies (kcat/Km) of wild-type BjPutA and R51E
are within a factor of 2 (Table 5). These results suggest
that tetramerization is not essential for the in vitro cat-
alytic activity of BjPutA.
We considered the hypothesis that dimeric R51E
assembles into the tetramer under the conditions of
the activity assay, i.e., that reduction of the FAD or
substrate binding enhances the association of two
dimers into the tetramer. To test this idea, we first per-
formed sedimentation velocity experiments after inacti-
vation by NPPG. NPPG locks the FAD into the
reduced state, thus mimicking a major effect of proline
oxidation on the enzyme. NPPG-inactivated R51E
displayed an apparent sedimentation coefficient of 5.9
S (Fig. 10C), which is close to that of untreated R51E
(6.5 S) and far from that of wild-type BjPutA (10.8 S).
We next performed sedimentation velocity of R51E in
the presence of the active site ligands THFA and
NAD+, which bind in the PRODH and GSALDH
active sites, respectively. We note that the binding of
these ligands to a monomeric type B PutA (CfPutA)
induces dimerization [25]. R51E in the presence of
THFA and NAD+ exhibits an apparent sedimentation
coefficient (6.2 S) consistent with a dimer (Fig. 10C).
Thus, in contrast to monomeric type B PutA, the
binding of active site ligands does not induce higher
order assembly of R51E. Finally, we conducted the
coupled activity assay for R51E as a function the
enzyme concentration. The observed rate is linearly
proportional to R51E concentration, as expected for
an enzyme that does not require assembly into a
higher order oligomer for activity (Fig. 10D). Taken
together, our results suggest that R51E forms a cat-
alytically competent dimer.
Discussion
Almost 50% of proteins are homooligomers, implying
that self-association underlies function [39,40]. There
are many possible reasons for oligomerization. For
example, substrate and cofactor binding sites of
Table 4. Structural and molecular mass (Mr) parameters from SAXS for BjPutA and BjPutA R51E.
BjPutA 2.3 mg�mL�1 4.7 mg�mL�1 7.0 mg�mL�1
Rg from Guinier (�A) 53 � 2 52 � 1 51.9 � 0.7
I(0) from Guinier (�A�1) 183 � 8 409 � 7 607 � 7
Dmax (�A) 140–146 140–146 136–140
Rg from P(r) (�A)a 51.4–51.6 51.6–51.7 51.4–51.5
I(0) from P(r) (�A�1)a 177–180 406–412 612–613
Porod volume (�A3)a 541 000–549 000 553 000–561 000 560 000
Volume of correlation, Vc (�A2)b 1577 1577 1566
Mr from Vc (kDa)b 380 390 380
Mr from MoW2 (kDa)c 426 424 426
Monomeric Mr from sequence 107 107 107
BjPutA R51E 2.3 mg�mL�1 4.7 mg�mL�1 7.0 mg�mL�1
Rg from Guinier (�A) 44.9 � 0.7 45.0 � 0.6 44.9 � 0.4
I(0) from Guinier (�A�1) 118.9 � 0.4 232 � 3 356 � 3
Dmax (�A) 140–150 136–145 140–150
Rg from P(r) (�A)a 45.3–45.7 44.9–45.0 45.8–46.0
I(0) from P(r) (�A�1)a 116–117 224–227 356–358
Porod volume (�A3)a 280 000–281 000 278 000–283 000 288 000–289 000
Volume of correlation, Vc (�A2)b 1005 1011 1010
Mr from Vc (kDa)b 180 180 180
Mr from MoW2 (kDa)c 231 234 219
Monomeric Mr from sequence 107 107 107
a From calculations of P(r) using PRIMUS [64] with the indicated range of Dmax.b Calculated with SCATTER 3.0 [70]. c Calculated from the
method of Fischer et al. [32].
3040 The FEBS Journal 284 (2017) 3029–3049 ª 2017 Federation of European Biochemical Societies
Conservation of a core dimer in type A PutAs D. A. Korasick et al.
enzymes can occur in oligomer interfaces [41,42]. Even
when the active site is not in an interface, oligomeriza-
tion may still be essential for catalytic activity, as in
some ALDHs [43,44]. Many integral membrane trans-
porters function as oligomers [45]. Quaternary structure
is also important in allosteric proteins [46], cooperative
enzymes [47], and morpheein enzymes [48]. Oligomeriza-
tion can also contribute to protein stability [41].
Here, we investigated the functional significance of
oligomerization in type A PutAs. The first type A
PutA studied, BjPutA, was found to be a tetramer
consisting of a pair of domain-swapped dimers that
form a ring [20]. Herein, we showed that several other
type A PutAs are dimeric, implying the BjPutA tetra-
mer may be an exception.
The domain-swapped dimer observed in all type A
PutA crystal structures, whether as a stand-alone
dimer or half of a tetramer, has obvious functional rel-
evance. The oligomerization flap of one protomer seals
the substrate-channeling tunnel of the other protomer,
as shown for BbPutA (Fig. 3C,D). Without this inter-
molecular lid, the intermediate would have a higher
probability of diffusing into the bulk medium. Thus,
domain-swapped dimerization in type A PutAs enables
the substrate-channeling step of the catalytic mecha-
nism. We note that in type B PutAs, the intermolecu-
lar lid is replaced by an intramolecular (tertiary
structural) interaction involving the C-terminal ALDH
superfamily domain [21,24,25].
The functional significance of the BjPutA tetramer is
not obvious, prompting the work reported here. The
dimer–dimer interface is formed by the N-terminal
arm and a-domain, two regions of the protein that
lack catalytic residues and ligand-binding sites. The
interface is far from the active sites and substrate-
channeling tunnel. For example, the hot spot residue
Arg51 is 30 �A from the nearest flavin N5 and 50 �A
from the catalytic Cys of the GSALDH site. Consis-
tent with these observations, the dimeric hot spot vari-
ant R51E exhibits wild-type catalytic behavior and
displays an apparent sedimentation coefficient consis-
tent with the formation of a dimer in solution. The
sedimentation data for R51E contain no evidence for
tetramer formation. Further, it was observed that nei-
ther flavin reduction nor incubation with active site
ligands induces tetramer formation by R51E or
BbPutA (Figs 5E and 10C). Therefore, it is concluded
that tetramerization is not essential for catalytic
function, suggesting that the domain-swapped dimer is
the core oligomeric structure of type A PutAs. We
note that this conclusion is rendered from in vitro bio-
chemical and biophysical analysis of PutAs and that,
under cellular conditions, molecular crowding or other
favorable conditions may promote higher-order
oligomerization.
Our results for BjPutA are consistent with the hot
spot theory of protein–protein interaction [49,50]. Hot
spots refer to the region of a protein–protein interface
that contains a few critical residues that account for
most of the association energy. Hot spots have a dis-
tinctive amino acid composition – often Trp, Arg, or
Tyr [50]. Consistent with the hot spot theory, we could
abrogate tetramerization of BjPutA with the single
mutation of Arg51 to Glu. It is interesting that this
Fig. 9. Comparison of the in-solution properties of BjPutA and
BjPutA R51E. (A) A representative experimental SAXS curve of
BjPutA R51E (4.7 mg�mL�1) is shown in open circles. Overlaid are
the theoretical SAXS curves calculated from the BjPutA domain-
swapped dimer (solid red) and the BjPutA tetramer (dashed red).
The v values obtained from FoXS fits of the theoretical scattering
curve of the crystallographic dimer and crystallographic tetramer to
the experimental scattering data are 1.3 and 51.9, respectively. (B)
Experimental distance distributions for wild-type BjPutA (black) and
R51E (red).
3041The FEBS Journal 284 (2017) 3029–3049 ª 2017 Federation of European Biochemical Societies
D. A. Korasick et al. Conservation of a core dimer in type A PutAs
particular Arg residue makes no specific electrostatic
interactions in the native tetramer (Fig. 7). Further-
more, Arg51 is located on a disordered loop. Thus,
visual inspection of the crystal structure might not
have revealed Arg51 as a hot spot residue. However,
analysis of the interface with PDBePISA indicated that
Arg51 contributes significantly to the dimer–dimer
interface surface area, despite the lack of the tradi-
tional electrostatic interactions one typically associates
with Arg. This result points to buried surface area as a
key metric for identifying hot spot residues.
In addition, our results indicate that the prediction
of quaternary structure and oligomeric state from crys-
tal packing remains challenging in some cases. The
Fig. 10. Kinetic data for wild-type BjPutA and R51E, and the effects of active site ligands on the oligomeric state of R51E. (A) Dependence
of the coupled PRODH-GSALDH reaction rate on proline concentration for wild-type BjPutA (black points). Kinetic data were fit to a
substrate inhibition model (red curve). (B) Dependence of the coupled PRODH-GSALDH reaction rate on proline concentration for R51E
(black points). Kinetic data were fit to a substrate inhibition model (red curve). The assays used fixed concentrations of proline (40 mM),
CoQ1 (0.1 mM), and NAD+ (0.2 mM). (C) The distribution of sedimentation coefficients observed from sedimentation velocity experiments of
R51E performed in the absence of ligands (solid black), after inactivation with NPPG (red), or in the presence of the active site ligands THFA
(10 mM) and NAD+ (1 mM) (blue). The distribution of sedimentation coefficients observed for wild-type BjPutA (dashed black) in the absence
of ligands is provided for reference. The enzyme concentration was 28 lM in all experiments. (D) Dependence of the coupled PRODH-
GSALDH reaction rate on protein concentration for R51E (black points). Data were fit to a linear regression model (red line).
Table 5. Kinetic Parameters for BjPutA and BjPutA R51E.
Km
(mM)
kcat
(s�1)
kcat/Km
(M�1�s�1) Ki (mM)
BjPutA 4 � 1 0.31 � 0.03 70 � 20 400 � 100
BjPutA R51E 10 � 2 0.40 � 0.04 40 � 10 220 � 50
3042 The FEBS Journal 284 (2017) 3029–3049 ª 2017 Federation of European Biochemical Societies
Conservation of a core dimer in type A PutAs D. A. Korasick et al.
PISA algorithm, which is based on physical–chemical
models of protein interactions and chemical thermody-
namics, is perhaps the gold standard for predicting oli-
gomeric state from crystal packing and is used by the
PDB to identify the most likely biological assembly
[33]. PISA analysis suggested that BbPutA forms a
stable tetramer in solution (Fig. 5D). However, our
SAXS and sedimentation data showed no evidence of
a tetramer at concentrations in the range of 1.8–4 mg�mL�1 (16–37 lM). Although it is possible that
the tetramer could form at even higher concentrations,
we have observed solubility problems with BbPutA at
concentrations above 10 mg�mL�1. It is possible that
the high protein concentration achieved as the crystal-
lization drop equilibrates promotes formation of the
higher order assembly seen in the crystal. Such high
concentrations are not possible in solution for
BbPutA, so the tetramer may not be observable with
standard solution biophysical techniques. Finally, it
remains possible that the tetramer could form in the
cell, where molecular crowding may enhance associa-
tion of dimers. We have also encountered this phe-
nomenon – where the crystal and solution data
conflict – with monofunctional GSALDHs [51]. In that
case, the wild-type enzyme is hexameric both in solu-
tion and in the crystal. However, mutation of a hex-
amerization hot spot residue produced dimeric protein
in solution. Curiously, the dimeric protein was
observed to form the hexamer in the crystal. These
results demonstrate that it remains challenging to pre-
dict oligomeric state in solution from crystal structures
for some systems.
Materials and methods
Production and crystallization of BbPutA
A synthetic gene encoding PutA from Bd. bacteriovorus
HD100 (BbPutA, 982 residues, NCBI RefSeq number NP_
968157.1) with codons optimized for expression in E. coli
was purchased from BIO BASIC Inc. (Markham, Ontario
CA). The gene was subcloned from pUC57 into pKA8H
between NdeI and BamHI sites. The encoded protein has
an N-terminal 8xHis tag that is cleavable by tobacco etch
virus protease (TEVP).
BbPutA was expressed in BL21-AI cells and purified
with affinity chromatography (Ni2+-charged HisTRAP;
GE Healthcare Life Sciences, Pittsburgh, PA, USA) using
protocols described previously for GsPutA [23]. The His
tag was cleaved as described previously for GsPutA [23].
Purified BbPutA was dialyzed into a storage buffer consist-
ing of 50 mM Tris, 125 mM NaCl, 1 mM EDTA, and 1 mM
tris(3-hydroxypropyl)phosphine (THP) at pH 7.5, and then
concentrated to 4 mg�mL�1 using stirred ultrafiltration.
The protein concentration was estimated using the bicin-
choninic acid assay (BCA).
Crystal screening and optimization trials of active
BbPutA produced weakly diffracting crystals, which were
unsuitable for structure determination. Therefore, we pur-
sued crystallization of BbPutA inactivated by the mecha-
nism-based inactivator NPPG (Fig. 4A). Previous studies
have shown that NPPG covalently modifies the FAD and
induces a conformation that resembles the 2-electron
reduced enzyme [23,28,29]. We hypothesized that the
change of conformation might enhance the crystallization
properties of BbPutA. This strategy produced crystals that
diffracted to 2.2 �A resolution.
BbPutA was incubated with NPPG (gift from C. Whit-
man) at a ratio of 1 mg of enzyme per 1 mg of inactivator
for 30 min on ice. The inactivated BbPutA was passed
through a 0.22 lM centrifugal filter device at 4 °C to
remove precipitate. Crystal screening trials were performed
at 20 °C with commercial kits using the microbatch method
with drops formed by mixing 1.5 lL of inactivated BbPutA
(3 mg�mL�1) and 1.5 lL of crystallization reagent. The
drops were covered with Al’s oil (Hampton Research, Aliso
Viejo, CA, USA). Several conditions yielded crystals over-
night. Based on the size and ease of reproducibility, the hit
from reagent 10 of Wizard III (Emerald Biosystems, Bain-
bridge Island, WA, USA) was selected for additional opti-
mization. These efforts produced crystals grown in the
presence of 19% (w/v) polyethylene glycol (PEG) 3350 and
0.25 M KSCN. This crystal form was improved by using
Hampton Index reagents as additives. The base condition
(19% (w/v) PEG 3350, 0.25 M KSCN) was mixed with each
Hampton Index reagent at a ratio of 80 : 20 (base : addi-
tive) and used in microbatch trials. Crystals of equivalent
quality were obtained using Index reagents 45, 72, 73, 76,
79 or 83 as additives. In preparation for cryogenic data col-
lection, the crystals were cryoprotected using 24% (w/v)
PEG 3350, 0.25 M KSCN, and 25% (v/v) ethylene glycol
and plunged into liquid nitrogen. The space group is
P21221 with the unit cell parameters listed in Table 1. The
asymmetric unit contains four protein molecules arranged
as two dimers. The method of Matthews predicts 57% sol-
vent (VM = 2.9 �A3�Da�1) [52].
X-ray diffraction data collection, phasing, and
refinement
Crystals of inactivated BbPutA were analyzed at Advanced
Photon Source beamline 24-ID-E. The data set used for
refinement consisted of 400 frames of data collected with
an oscillation width of 0.25° and detector distance of
300 mm. The data were processed with XDS [53] and SCALA
[54]. Initial phases were determined with molecular replace-
ment as implemented in MOLREP [55] using a search model
derived from the coordinates of GsPutA (47% sequence
3043The FEBS Journal 284 (2017) 3029–3049 ª 2017 Federation of European Biochemical Societies
D. A. Korasick et al. Conservation of a core dimer in type A PutAs
identity, Table 2). The structure was refined in PHENIX [56]
and adjusted manually with COOT [57]. The refinement cal-
culations used noncrystallographic symmetry restraints.
The B-factor model consisted of one TLS group per protein
chain and isotropic B-factors for all nonhydrogen atoms.
The model was validated with MOLPROBITY [58,59].
Production of LpPutA and DvPutA
The gene encoding PutA from L. pneumophila subsp. pneu-
mophila (LpPutA, 1054 residues, NCBI RefSeq WP_
010947423.1) was cloned from genomic DNA purchased
from ATCC (catalog number 33152D) and inserted into
pET151. The expressed protein contains an N-terminal
6xHis tag followed the V5 epitope and TEVP cleavage site.
LpPutA was expressed in BL21 DE3 Star cells and purified
with affinity chromatography (Ni2+-charged HisTRAP) and
anion exchange chromatography (HiTrap Q; GE Health-
care) using protocols similar to those described for BjPutA
[60]. The His tag was cleaved as described for BjPutA [60].
The purified protein was dialyzed overnight at 4 °C into a
buffer containing 50 mM Tris-HCl, 50 mM NaCl, 0.5 mM
THP, 5% (v/v) glycerol, and 0.5 mM EDTA at pH 7.5.
The gene encoding PutA from D. vulgaris str. Hildenbor-
ough (DvPutA, 1006 residues, NCBI RefSeq WP_
010940575.1) in the expression plasmid pNIC28-Bsa4 was
obtained from the New York Structural Genomics
Research Consortium. The expressed protein has an N-
terminal 6xHis tag and TEVP cleavage site. DvPutA was
expressed in BL21(DE3)pLysS and purified as described
above for LpPutA. The His tag was not cleaved. The puri-
fied protein was dialyzed overnight at 4 °C into a storage
buffer containing 50 mM Tris-HCl, 50 mM NaCl, 0.5 mM
EDTA, 5% (v/v) glycerol, and 0.5 mM THP at pH 7.5.
Mutagenesis, production, and activity assays of
BjPutA and BjPutA R51E
Wild-type and mutant PutA from Br. japonicum (BjPutA)
in the pKA8H vector were expressed and purified as previ-
ously described [60]. The His tag was removed from both
enzymes as previously described [60]. The R51E mutant
variant of BjPutA was generated from the pKA8H-BjPutA
construct using the QuikChange II XL kit (Agilent, Santa
Clara, CA, USA). The purified proteins were dialyzed over-
night against a storage buffer containing 50 mM Tris (pH
7.8), 50 mM NaCl, 0.5 mM Tris(2-carboxyethyl)phosphine,
and 5% (v/v) glycerol.
Assessment of the coupled PRODH-GSALDH activity
of both wild-type and R51E BjPutA was carried out as pre-
viously described [26]. Briefly, NADH formation was moni-
tored at 340 nm in assays performed at room temperature
in an Epoch 2 plate reader (BioTek, Winooski, VT, USA).
The reaction mixture contained BjPutA or BjPutA R51E
(0.5 lM), proline (2.5–400 mM), coenzyme Q1 (0.1 mM), and
NAD+ (0.2 mM) in a buffer containing 50 mM potassium
phosphate, 25 mM NaCl, and 10 mM MgCl2 at pH 7.5 with
a final reaction volume of 200 lL. Reactions were per-
formed in the presence and absence of NAD+ to correct
for CoQ1 reduction. Linear regression in Origin 2017 was
used to determine rates for final analysis. The rate data
were fit to a substrate inhibition model in Origin 2017.
Analytical ultracentrifugation
Sedimentation velocity experiments were performed in a
Beckman XL-I analytical ultracentrifuge using an An50Ti
rotor at 20 °C. Aliquots of the protein solution and dialysis
buffer (reference buffer) were loaded into a sedimentation
velocity cell, bearing a two-sector charcoal-Epon center-
piece. Prior to centrifugation, the sample was allowed to
equilibrate for 2 h. The sample was then centrifuged at
82 021 g for 300 radial scans at 2-min intervals acquired
using Rayleigh interference optics. Scans 10–300 were used
in the analysis. Sedimentation coefficient, c(s), and molecular
mass, c(M), distributions were generated using SEDFIT [61].
Prior to sedimentation analysis of NPPG-inactivated
BbPutA and BjPutA R51E, each protein was treated with
1 mg NPPG (BOC Sciences, Shirley, NY, USA) per 1 mg
of protein, incubated on ice for 30 min, and then loaded
into the sedimentation cell. The reference for sedimentation
was the respective storage buffer (listed in the purification
protocols above) supplemented with an equal amount of
NPPG used in the inactivation step.
Prior to sedimentation analysis of BbPutA and BjPutA
R51E in the presence of active site ligands, protein samples
were supplemented with 10 mM THFA and 1 mM NAD+
and dialyzed for 4 h with two buffer exchanges in a
Slide-a-lyzer mini-dialysis device (ThermoFisher, Waltham,
MA, USA) against the storage buffer (listed in the purifica-
tion protocol above) supplemented with 10 mM THFA and
1 mM NAD+. The dialysate served as the reference for sed-
imentation.
For all BjPutA wild-type and mutant samples, the fric-
tional ratio was allowed to vary during global fitting. In
the analysis of BbPutA, the frictional ratio was set at 1.75
to account for sample aggregation and precipitation. We
note that the frictional ratio of BjPutA R51E was approxi-
mately 1.75, which is why this frictional ratio was applied
to the BbPutA data.
SAXS
Purified BbPutA was prepared for SAXS by passing it
through a prepacked Superdex-200 10 9 300 mm size
exclusion chromatography (SEC) column (GE Life
Sciences, Pittsburgh, PA, USA) equilibrated with 50 mM
Tris, 125 mM NaCl, 1 mM EDTA, and 1 mM THP at pH
7.5 at a flow rate of 0.5 mL�min�1. The protein eluted as a
single peak that was baseline separated from the void
3044 The FEBS Journal 284 (2017) 3029–3049 ª 2017 Federation of European Biochemical Societies
Conservation of a core dimer in type A PutAs D. A. Korasick et al.
volume of the column (Fig. 11). Fractions under the peak
were pooled for SAXS analysis. A sample of the SEC flow-
through was reserved for use in the SAXS background
measurement.
Purified LpPutA and DvPutA were prepared for SAXS
by passing them through the aforementioned SEC column
equilibrated with 50 mM Tris, 500 mM NaCl, and 1 mM
THP at pH 8.0 (flow rate of 0.5 mL�min�1). Each protein
eluted as a single peak that was baseline separated from
the void volume. The protein in the single peak was pooled
and concentrated using a centrifugal concentration device
(30-kDa cutoff) to 8 mg�mL�1 as monitored by the BCA
assay. The SEC equilibration buffer was used for the SAXS
background measurement.
Purified wild-type BjPutA and R51E were prepared for
SAXS by passing them through the aforementioned SEC
column equilibrated with 50 mM Tris (pH 7.8), 50 mM NaCl,
0.5 mM Tris(2-carboxyethyl)phosphine, and 5% (v/v) glyc-
erol (flow rate of 0.5 mL�min�1). Each protein eluted as a
single peak that was baseline separated from the void volume
(Fig. 11). The protein in the single peak was pooled and con-
centrated using a centrifugal concentration device (50-kDa
cutoff). The concentrated protein was dialyzed overnight
using the SEC buffer. The final protein concentration after
dialysis was 7 mg�mL�1 (BCA method). A sample of the dia-
lysate was reserved for the SAXS background measurement.
The protein samples were transferred to 96-well plates
and shipped at 4 °C to the SIBYLS beamline 12.3.1 of the
Advanced Light Source through the mail-in program
[62,63]. Additional SEC steps were not performed at the
beamline. The SAXS intensity data – I(q) vs. q, q = 4psinh/k, where 2h is the scattering angle and k is the X-ray
wavelength in angstroms – were measured at three nominal
protein concentrations in the range of 1–8 mg�mL�1. Note
that scattering intensities for BbPutA were calculated only
at a single protein concentration, 1.8 mg�mL�1. Because of
the low concentration of the BbPutA SEC fractions, data
were collected at a single concentration of 1.8 mg�mL�1.
For BbPutA, DvPutA, and LpPutA, data were collected
for each protein concentration at exposure times of 0.5,
1.0, 3.0, and 6.0 s. For both wild-type BjPutA and the
BjPutA R51E mutant variant, data were collected in shut-
terless mode using a Pilatus detector with a total of 33
evenly spaced images acquired over 10.2 s (0.3 s�frame�1).
The scattering curves collected from the protein samples
were corrected for background scattering using intensity
data collected from the aforementioned reference buffers.
For all except the BjPutA and BjPutA R51E samples,
composite scattering curves were generated with PRIMUS [64]
by scaling and merging the background-corrected low q
region data from the 0.5 s exposure with the high q region
data from the 3.0 s exposure. For both wild-type BjPutA
and BjPutA R51E, composite scattering curves were gener-
ated with PRIMUS by averaging and merging the back-
ground-corrected low q region data from the first three
(0.9 s) exposures with the high q region data from the first
12 (3.6 s) exposures.
The composite SAXS curves were analyzed as follows.
PRIMUS was used to perform Guinier analysis. GNOM was
used to calculate distance distribution functions [65]. FoXS
and MultiFoXS [35] were used to calculate theoretical
SAXS curves from atomic models. For BjPutA and
BbPutA, crystal structures were input to FoXS. For
DvPutA and LpPutA, homology models were generated
using SWISS-MODEL [66], RaptorX [67], and Phyre2 [68].
The LpPutA model was improved with AllosMod-FoXS
[34,69], which added missing residues and allowed for inter-
domain movements. The molecular mass in solution was
determined from SAXS data using the volume of correla-
tion invariant [31] as implemented in Scatter 3.0 [70] and
with the SAXS MoW2 server [32].
DAMMIF [36] was used for shape reconstructions. For
each reconstruction, 50 independent calculations were per-
formed. Two-fold symmetry was enforced during the recon-
structions of the dimeric PutAs (BbPutA, DvPutA,
LpPutA, and BjPutA R51E). Point group 222 symmetry
was enforced during the shape reconstruction of wild-type
BjPutA. The models from DAMMIF were averaged and fil-
tered with DAMAVER [71]. The averaged and filtered dummy
atom models (dammif.pdb) were superimposed onto crystal
structures or homology models with SUPCOMB [72]. The
pdb2vol utility of situs [73] was used to convert dummy
atom models (dammif.pdb) into volumetric maps. The col-
ores utility of situs was used to calculate the correlation
coefficient between atomic models and volumetric maps.
The SAXS data have been deposited in the SASBDB [74]
under the following accession codes: SASDCP3 (BbPutA),
Fig. 11. SEC chromatograms for BbPutA (blue), wild-type BjPutA
(black), and BjPutA R51E (red) obtained with a prepacked
Superdex-200 10 9 300 mm SEC column (GE Life Sciences)
connected to an AKTA pure chromatography instrument (GE Life
Sciences).
3045The FEBS Journal 284 (2017) 3029–3049 ª 2017 Federation of European Biochemical Societies
D. A. Korasick et al. Conservation of a core dimer in type A PutAs
SASDCQ3 (DvPutA 1.5 mg�mL�1), SASDCX3 (DvPutA
3.0 mg�mL�1), SASDCY3 (DvPutA 4.5 mg�mL�1),
SASDCR3 (LpPutA 3.0 mg�mL�1), SASDCV3 (LpPutA
5.0 mg�mL�1), SASDCW3 (LpPutA 8.0 mg�mL�1),
SASDCS3 (BjPutA 2.3 mg�mL�1), SASDCT3 (BjPutA
4.7 mg�mL�1), SASDCU3 (BjPutA 7.0 mg�mL�1),
SASDCZ3 (R51E 2.3 mg�mL�1), SASDC24 (R51E
4.7 mg�mL�1), SASDC34 (R51E 7.0 mg�mL�1),
Acknowledgements
Research reported in this publication was supported
by the NIGMS of the National Institutes of Health
under award number R01GM065546. We thank
Kevin Dyer and Katherine Burnett for collecting
SAXS data through the SIBYLS mail-in program.
We thank Jonathon Schuermann for help with X-ray
diffraction data collection and processing. We thank
Chris Whitman and William Johnson, Jr. for provid-
ing the NPPG that was used for crystallization of
BbPutA. We thank Prof Steven Almo and the New
York Structural Genomics Consortium for providing
the DvPutA plasmid. We thank Dina Schneidman for
help with running the AllosMod-FoXS server. Part of
this research was performed at the Advanced Light
Source. The Advanced Light Source is supported by
the Director, Office of Science, Office of Basic Energy
Sciences, of the U.S. Department of Energy under
contract no. DE-AC02-05CH11231. Additional sup-
port for the SYBILS beamline comes from the
National Institute of Health project MINOS
(R01GM105404). Part of this work is based on
research conducted at the Northeastern Collaborative
Access Team beamlines, which are funded by the
National Institute of General Medical Sciences from
the National Institutes of Health (P41 GM103403).
This research used resources of the Advanced Photon
Source, a U.S. Department of Energy (DOE) Office
of Science User Facility operated for the DOE Office
of Science by Argonne National Laboratory under
contract no. DE-AC02-06CH11357.
Conflict of interest
The authors declare that they have no conflicts of
interest with the contents of this manuscript.
Author contributions
DAK, HS, and JJT designed experiments; DAK, HS,
and TAP performed experiments; DAK, HS, and JJT
analyzed data; ML and RD contributed reagents;
DAK and JJT wrote the article.
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