Crystal structures and kinetics of monofunctional proline dehydrogenase provide insight into substrate recognition and conformational changes associated with flavin reduction and product release

Article abstract of DOI:10.1021/bi301312f

Proline dehydrogenase (PRODH) catalyzes the FAD-dependent oxidation of proline to Δ¹-pyrroline-5-carboxylate, which is the first step of proline catabolism. Here, we report the structures of proline dehydrogenase from Deinococcus radiodurans in the oxidized state complexed with the proline analogue l-tetrahydrofuroic acid and in the reduced state with the proline site vacant. The analogue binds against the si face of the FAD isoalloxazine and is protected from bulk solvent by helix α8 and the β1-α1 loop. The FAD ribityl chain adopts two conformations in the E-S complex, which is unprecedented for flavoenzymes. One of the conformations is novel for the PRODH superfamily and may contribute to the low substrate affinity of Deinococcus PRODH. Reduction of the crystalline enzyme-inhibitor complex causes profound structural changes, including 20 butterfly bending of the isoalloxazine, crankshaft rotation of the ribityl, shifting of α8 by 1.7 A?, reconfiguration of the β1-α1 loop, and rupture of the Arg291-Glu64 ion pair. These changes dramatically open the active site to facilitate product release and allow electron acceptors access to the reduced flavin. The structures suggest that the ion pair, which is conserved in the PRODH superfamily, functions as the active site gate. Mutagenesis of Glu64 to Ala decreases the catalytic efficiency 27-fold, which demonstrates the importance of the gate. Mutation of Gly63 decreases the efficiency 140-fold, which suggests that flexibility of the β1-α1 loop is essential for optimal catalysis. The large conformational changes that are required to form the E-S complex suggest that conformational selection plays a role in substrate recognition.

Full text of DOI:10.1021/bi301312f

Article
pubs.acs.org/biochemistry
Crystal Structures and Kinetics of Monofunctional Proline
Dehydrogenase Provide Insight into Substrate Recognition and
Conformational Changes Associated with Flavin Reduction and
Product Release
Min Luo,† Benjamin W. Arentson,‡ Dhiraj Srivastava,† Donald F. Becker,‡ and John J. Tanner*,†,§
†Department of Chemistry, University of MissouriColumbia, Columbia, Missouri 65211, United States
‡Department of Biochemistry, University of NebraskaLincoln, Lincoln, Nebraska 68588, United States
§Department of Biochemistry, University of MissouriColumbia, Columbia, Missouri 65211, United States
S
* Supporting Information
ABSTRACT: Proline dehydrogenase (PRODH) catalyzes the
FAD-dependent oxidation of proline to Δ1-pyrroline-5-
carboxylate, which is the first step of proline catabolism.
Here, we report the structures of proline dehydrogenase from
Deinococcus radiodurans in the oxidized state complexed with
the proline analogue L-tetrahydrofuroic acid and in the reduced
state with the proline site vacant. The analogue binds against
the si face of the FAD isoalloxazine and is protected from bulk
solvent by helix α8 and the β1−α1 loop. The FAD ribityl chain
adopts two conformations in the E−S complex, which is
unprecedented for flavoenzymes. One of the conformations is novel for the PRODH superfamily and may contribute to the low
substrate affinity of Deinococcus PRODH. Reduction of the crystalline enzyme−inhibitor complex causes profound structural
changes, including 20° butterfly bending of the isoalloxazine, crankshaft rotation of the ribityl, shifting of α8 by 1.7 Å,
reconfiguration of the β1−α1 loop, and rupture of the Arg291−Glu64 ion pair. These changes dramatically open the active site
to facilitate product release and allow electron acceptors access to the reduced flavin. The structures suggest that the ion pair,
which is conserved in the PRODH superfamily, functions as the active site gate. Mutagenesis of Glu64 to Ala decreases the
catalytic efficiency 27-fold, which demonstrates the importance of the gate. Mutation of Gly63 decreases the efficiency 140-fold,
which suggests that flexibility of the β1−α1 loop is essential for optimal catalysis. The large conformational changes that are
required to form the E−S complex suggest that conformational selection plays a role in substrate recognition.
roline dehydrogenase (PRODH) catalyzes the first
suppressor protein.5−13 POX expression is induced by tumor
suppressor p53, and POX itself activates intrinsic and extrinsic
apoptotic pathways.8 Crucial to the role of POX as a tumor
suppressor is its ability to generate superoxide.5,8,14 Also, certain
mutations in the gene encoding POX cause type I hyper-
prolinemia,15 which is a risk factor for schizophrenia.16
Crystal structures of PRODHs suggest that conformational
changes are essential for catalysis, but the details of these
conformational changes have not been elucidated. We
previously determined structures of the PRODH domain of
Escherichia coli PutA (PutA86−669) complexed with proline
and proline analogues,17−19 as well as a structure of
Bradyrhizobium japonicum PutA with a sulfate ion bound in
the proline site.20 In all of those structures, proline (or the
analogue) is completely buried, implying that the active site
opens to allow product release and closes again in response to
P
reaction of proline catabolism (Figure 1A).1 PRODH is
an FAD-dependent enzyme that catalyzes the oxidation of L-
proline to Δ1-pyrroline-5-carboxylate (P5C). The electrons
stored in the reduced flavin are subsequently transferred to the
electron transport chain for eventual ATP production. P5C
forms an equilibrium with its hydrolysis product glutamate γ-
semialdehyde (GSA), which is oxidized to glutamate by the
second enzyme of proline catabolism, NAD+-dependent P5C
dehydrogenase (P5CDH). PRODH and P5CDH are distinct
enzymes in eukaryotes and Gram-positive bacteria, whereas the
two enzymatic activities are combined into a single polypeptide
chain, known as proline utilization A (PutA), in Gram-negative
bacteria.2,3
PRODH is of interest because of its roles in apoptosis,
cancer, and schizophrenia. In eukaryotes, PRODH and P5CDH
are localized to the inner mitochondrial membrane and
mitochondrial matrix, respectively, and proline catabolism is
important for establishing the mitochondrial redox status.4,5
Seminal work from Phang’s group has established that human
PRODH (also known as proline oxidase or POX) is a tumor
Received: September 26, 2012
Revised: November 10, 2012
Published: November 14, 2012
© 2012 American Chemical Society
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Figure 1. (A) Reactions catalyzed by PRODH and P5CDH. (B) Scheme showing the enzyme states that occur during the PRODH reductive half-
reaction.
substrate binding. Because the structure of the empty PutA
PRODH active site is unknown, it has not been possible to
deduce these conformational changes for PutA. We also
reported a structure of the monofunctional PRODH from
Thermus thermophilus (TtPRODH) in a substrate-free con-
formation.21 However, the corresponding structure of the
closed, substrate-bound active site is unknown. Thus, our
understanding of how conformational changes facilitate
catalysis has been limited by difficulties in crystallizing the
same PRODH in both the open (substrate-free) and closed
(substrate-bound) conformations. Although some information
has been gained by comparing the structures of PutA669-
proline and ligand-free TtPRODH, this comparison is
problematic because it is unknown whether PutAs and
monofunctional PRODHs bind proline similarly. Another
factor that complicates such a comparison is that the
PRODH domain of PutA makes tertiary structural contacts
that are absent in monofunctional PRODHs, and thus, it is
unlikely that PutA PRODH domains and monofunctional
PRODHs exhibit similar degrees of flexibility.
We therefore surveyed several monofunctional PRODHs in
search of one that crystallizes in the presence of the proline
analogue L-tetrahydrofuroic acid (THFA). This survey
uncovered Deinococcus radiodurans R1 PRODH (DrPRODH)
as a suitable enzyme for determining the structural changes that
occur in monofunctional PRODHs during the reductive half-
reaction (Figure 1B). Herein, we report high-resolution crystal
structures and kinetic studies of DrPRODH, which provide
new insight into substrate recognition by monofunctional
PRODHs and the conformational changes that accompany
flavin reduction and product release.
DrPRODH and mutants G63A and E64A were expressed in
BL21(DE3)pLysS cells as follows. Starter cultures of 10 mL
were grown in LB medium overnight and used to inoculate 3 L
of LB broth. After the culture had reached an optical density
(OD600) of 0.8, 0.5 mM IPTG was added to induce protein
expression for 5 h at 22 °C. The cells were collected by
centrifugation, resuspended in 50 mM Tris, 100 mM NaCl, 10
mM imidazole, and 5% glycerol (pH 7.5), and frozen at −80
°C.
DrPRODH and mutants G63A and E64A were purified as
follows. The frozen cells were thawed at 4 °C in the presence of
protease inhibitors (0.1 mM TPCK, 0.05 mM AEBSF, 0.1 μM
pepstatin, 0.01 mM leupeptin, and 5 μM E-64) and broken
using sonication. The mixture was centrifuged at 16500 rpm in
an SS34 rotor for 1 h at 4 °C, filtered through a 0.45 μm filter
(Millipore), and loaded into a HisTrap HP column (5 mL) that
had been equilibrated in 50 mM Tris, 300 mM NaCl, 10 mM
imidazole, and 5% glycerol (pH 7.5). Washing steps with the
loading buffer supplemented with 10 mM imidazole followed
by 30 mM imidazole were performed. The protein was eluted
with 300 mM imidazole. The histidine tag was removed by
incubating the protein with 0.2 mg/mL TEVP for 1 h at 28 °C
followed by dialysis at 4 °C against 50 mM Tris, 50 mM NaCl,
and 5% glycerol (pH 7.5). The mixture was applied to the
HisTrap HP column to separate the cleaved protein, which
appeared in the flow-through, from the tag and TEVP. The
cleaved protein was dialyzed overnight in the dark at 4 °C into
50 mM Tris, 0.5 mM EDTA, 0.5 mM DTT, and 5% glycerol
(pH 7.8) in preparation for anion exchange chromatography
(HiTrap Q). The sample was loaded onto the column using a
buffer consisting of 50 mM Tris and 5% glycerol (pH 7.8), and
a linear NaCl gradient was applied. DrPRODH eluted in the
range of 360−420 mM NaCl. The protein concentration was
estimated using the bicinchoninic acid method (Pierce kit) with
bovine serum albumin as the standard. The flavin contents of
the purified mutants G63A and E64A were similar to that of
DrPRODH.
Crystallization and Crystal Soaking Experiments. All
crystallization experiments were performed at 22 °C using the
sitting-drop method of vapor diffusion with drops formed by
mixing equal volumes of the reservoir and the protein stock
solutions. The latter solution consisted of 2.8 mg/mL
DrPRODH in 50 mM Tris, 50 mM NaCl, 0.5 mM THP, 0.5
mM EDTA, 5% glycerol, and 400 mM THFA (pH 7.5). Initial
conditions were identified using commercially available crystal
screens (Hampton Research). Optimized crystals were grown
using a reservoir solution of 0.2 M MgCl2, 25% (w/v) PEG
3350, and 0.1 mM Bis-Tris (pH 5.8). The crystals were
cryoprotected with the reservoir solution supplemented with
25% (v/v) PEG 200, picked up with Hampton loops, and flash-
EXPERIMENTAL PROCEDURES
■
Protein Expression and Purification. The gene encoding
DrPRODH (NCBI RefSeq code NP_294538.1, 310 residues)
in the pMH1F vector was obtained from the Joint Center for
Structural Genomics. The gene was amplified by polymerase
chain reaction and ligated into plasmid pKA8H using NdeI and
BamHI sites. The expressed protein includes an N-terminal
His8 tag and tobacco etch virus protease (TEVP) site. The
G63A mutant of DrPRODH was created with the Quick-
Change II site-directed mutagenesis kit (Agilent) using a
forward primer (5′-GAACCTTGACCTGCTCGCCGAATTT-
ATCGACAGCCCG-3′) and a reverse primer (5′-CGGGCT-
GTCGATAAATTCCGCGAGCAGGTCAAGGTTC-3′). The
E64A mutant of DrPRODH was prepared similarly using a
forward primer (5′-CTTGACCTGCTCGGCGCCTTTATC-
GACAGCCCGGCC-3′) and a reverse primer (5′-GGCCGG-
GCTGTCGATAAAGGCGCCGAGCAGGTCAAG-3′). The
mutations were confirmed using DNA sequencing.
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a
cooled in liquid nitrogen. The space group is P212121 with the
following unit cell dimensions: a = 44 Å, b = 95 Å, and c = 136
Å. The asymmetric unit includes two enzyme−THFA
complexes and 40% solvent.
Crystals of DrPRODH with the FAD reduced (DrPRODHr)
were obtained by soaking the aforementioned crystals for ∼17
min in 50 mM sodium dithionite, 0.2 M MgCl2, 25% PEG
3350, 0.1 mM Bis-Tris (pH 5.5), and 25% PEG 200. The
crystal changed from deep yellow to colorless during soaking,
which is consistent with reduction of the FAD. The crystals
were flash-cooled in liquid nitrogen to trap the reduced enzyme
conformation.
X-ray Diffraction Data Collection, Processing, and
Refinement. Diffraction data were collected on beamlines
4.2.2 of the Advanced Light Source and 24-ID-C of the
Advanced Photon Source. The reflections were integrated with
XDS22 and scaled with SCALA23 (Table 1). The phase problem
was solved using molecular replacement as implemented in
MOLREP.24 The search model was the (βα)8 barrel of T.
thermophilus PRODH (residues 37−279 of chain A of Protein
Data Bank (PDB) entry 2G37, 47% identical to DrPRODH).
The correct solution had a correlation coefficient of 0.36 with
two molecules in the P212121 asymmetric unit. For reference,
molecular replacement calculations that assumed other
primitive orthorhombic space groups yielded correlation
coefficients of 0.26−0.31. The model from molecular
replacement was used as the starting point for several rounds
of model building with COOT25 and refinement with
PHENIX.26 Table 1 lists refinement statistics.
Table 1. X-ray Diffraction Data Collection and Refinement
DrPRODHo−THFA
oxidized
DrPRODHr
FAD redox state
space group
reduced
P212121
P212121
unit cell
parameters (Å)
a = 44.5, b = 95.5,
c = 136.4
a = 43.4, b = 95.7,
c = 136.0
wavelength (Å)
resolution (Å)
0.979
1.00
95.5−1.36 (1.43−1.36)
47.8−1.75 (1.84−1.75)
415890
no. of observations 450935
no. of unique
reflections
124272
58089
R
R
R
merge(I)
meas(I)
pim(I)
0.032 (0.431)
0.043 (0.576)
0.021 (0.294)
17.1 (2.4)
0.065 (0.837)
0.071 (0.905)
0.026 (0.341)
22.1 (2.5)
100.0 (100.0)
7.2 (7.0)
mean I/σ
completeness (%) 98.9 (98.5)
multiplicity
3.6 (3.7)
562
no. of protein
residues
550
no. of atoms
5091
4557
106
0
no. of FAD atoms 212
no. of THFA
atoms
16
no. of water
molecules
475
297
Rcryst
0.148 (0.239)
0.181 (0.279)
0.015
0.182 (0.276)
0.213 (0.323)
0.013
b
Rfree
rmsd for bond
lengths (Å)
c
rmsd for bond
angles (deg)
1.75
1.45
The oxidized flavin exhibits two conformations in the THFA
complex (Figure S1 of the Supporting Information). The two
conformations have occupancies of 0.56 (conformation A) and
0.44 (conformation B) and differ in the orientations of the 2′-
and 3′-hydroxyl groups of the ribityl chain. Justification for
building two conformations was obtained by refining the
structure with single conformations of the FAD. For example,
the Fo − Fc map calculated after a refinement that included only
conformation A at an occupancy of 1.0 showed strong features
for the 2′-OH and 3′-OH ribityl groups of conformation B
(Figure S1 of the Supporting Information, blue cage).
Conversely, the map calculated after a refinement that included
just conformation B at an occupancy of 1.0 showed strong
features for the 2′-OH and 3′-OH ribityl groups of
conformation A (Figure S1 of the Supporting Information,
green cage).
Enzyme Activity Assays. All chemicals used during kinetic
characterization were purchased from Fischer Scientific or
Sigma-Aldrich. All steady-state assays were conducted at 23 °C
in 50 mM potassium phosphate and 25 mM NaCl (pH 7.5).
For all assays, coenzyme Q1 (CoQ1) was used as the electron
acceptor with CoQ1 reduction monitored by the decrease in
absorbance at 278 nm using a molar extinction coefficient of
14.5 mM−1 cm−1.27 The Km for proline and the kcat for wild-
type DrPRODH (0.25 μM) were determined by varying the
concentration of proline (0−500 mM) while keeping the CoQ1
concentration constant (200 μM). Inhibition of wild-type
DrPRODH (0.25 μM) by THFA was analyzed by varying the
proline (10−500 mM) and THFA (0−200 mM) concen-
trations while keeping the CoQ1 concentration constant (200
μM). These assays were performed in a total volume of 200 μL
per assay using a Powerwave XS microplate spectrophotometer
(Bio-Tek). The Km for CoQ1 and the kcat for wild-type
DrPRODH (0.25 μM) were determined by varying the CoQ1
Ramachandran
d
plot
favored
(%)
99.3
0.7
99.3
0.7
allowed
(%)
outliers
(%)
average B (Å2)
protein
FAD
0.0
0.0
21.4
28.6
17.7
23.7
THFA
17.5
not applicable
26.2
acetate
not applicable
31.5
water
31.4
coordinate error
0.14
0.19
e
(Å)
PDB entry
4H6Q
4H6R
a
b
c
Values for the outer resolution shell of data are given in parentheses.
A common test set (5%) was used for refinement of both structures.
Compared to the parameters of Engh and Huber.34 The
d
Ramachandran plot was generated with RAMPAGE.35 Maximum
e
likelihood-based coordinate error estimate.
concentration (0−450 μM) and keeping the proline concen-
tration constant (500 mM). For the DrPRODH mutants G63A
(11 μM) and E64A (6.3 μM), the Km for proline and the kcat
were determined by varying the proline concentration (0−1000
mM) and holding the CoQ1 concentration constant (200 μM).
The Km for CoQ1 and the kcat for the DrPRODH mutants were
determined by varying the CoQ1 concentration (10−450 μM)
and holding the proline concentration constant (500 mM).
These assays were performed in a total volume of 150 μL by
mixing enzyme and substrate solutions using a Hi-Tech
Scientific SF-61DX2 stopped-flow instrument equipped with
a 0.15 cm path length cell. Steady-state parameters were
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Figure 2. Overall fold of PRODH. (A) Two views of the DrPRODHo−THFA structure. The backbone is colored according to a rainbow scheme,
with blue at the N-terminus and red at the C-terminus. FAD is colored yellow. THFA is colored pink. Residue numbers of the N- and C-termini are
noted. (B) Superposition of the DrPRODHo−THFA complex (yellow), DrPRODHr (gray), and TtPRODH (cyan, PDB entry 2g37). The R291−
E64 ion pair is observed only in the DrPRODHo−THFA structure. This figure and others were prepared with PyMOL.36
calculated by fitting initial rate data to the Michaelis−Menten
equation, and inhibition data were globally fit to a competitive
inhibition model using Enzyme Kinetic Wizard (SigmaPlot
12.0).
The oxidized flavin exhibits two conformations, which is
unprecedented for PRODHs and PutAs (Figure 4A).
Furthermore, we are unaware of another flavin oxidoreductase
that exhibits multiple flavin conformations in the E−S complex.
The two conformations have occupancies of 0.56 (conforma-
tion A) and 0.44 (conformation B). Interestingly, the two FAD
conformations do not correspond to THFA-bound and THFA-
free states because the occupancy of THFA is clearly 1.0 (B
factor = 17.5 Å2). Rather, the enzyme is able to bind THFA
using either of two different FAD conformations.
The two FAD conformations differ in the orientations of the
2′- and 3′-hydroxyl groups of the ribityl chain (Figure 4A and
Figure S1 of the Supporting Information). In conformation A
(yellow in Figure 4A), the 2′-OH points toward the proline
binding site while the 3′-OH is oriented 180° away and
interacts with the carbonyl of Gly191. Conformation B is
related to conformation A by a crankshaft rotation around the
C2′−C3′ bond. In conformation B (pink in Figure 4A), the 2′-
OH is tucked under the pyrimidine ring where it forms
hydrogen bonds with the flavin N1 atom and NH group of
Gly191, while the 3′-OH interacts with Glu295 of α8.
Ribityl conformation A appears to be unique among the
PRODH superfamily. Although it resembles the ribityl of the
PutA86−669−THFA complex in that the 2′-OH points into
the proline site, the other two hydroxyl groups of conformation
A are rotated 180° from the corresponding hydroxyls of the
PutA86−669−THFA complex (Figure 5A). Although the 4′-
OH of DrPRODH overlaps the 3′-OH of the PutA86−669−
THFA complex (Figure 5A), the two hydroxyls are non-
equivalent because they form different interactions. The 3′-OH
of the PutA86−669−THFA complex forms a hydrogen bond
RESULTS
■
Structure of the DrPRODHo−THFA Complex. The
crystal structure of oxidized DrPRODH (DrPRODHo)
complexed with the proline analogue THFA was determined
at 1.36 Å resolution (Table 1). It is the first structure of a
monofunctional PRODH complexed with a proline analogue
and the highest-resolution structure of any PRODH or PutA.
The DrPRODHo−THFA complex exhibits a distorted (βα)8
barrel fold in which the last helix, denoted α8 (residues 285−
295), is located above the C-terminal ends of the strands rather
than alongside β8 as in the classic triosephosphate isomerase
barrel (Figure 2A). This fold is also observed in the structures
of TtPRODH (Figure 2B) and PutA and is considered to be a
defining characteristic of the PRODH superfamily, which
includes both monofunctional PRODHs and PutAs. The root-
mean-square deviation between the DrPRODHo−THFA
complex and TtPRODH is 1.2 Å; the variation from PutA
PRODH domains is slightly higher, 1.5−1.9 Å.
THFA binds between the si face of the FAD and α8 (Figure
2A). Arg291, Arg292, and Lys98 form ion pairs with the
carboxylate of THFA, while Leu257, Tyr278, and Tyr288
provide hydrophobic contacts with the pyrrolidine ring (Figure
3A). Arg291 and Arg292 are stabilized by ion pairs with Glu64
and Glu295, respectively. The plethora of residues clustered
around THFA make the inhibitor and the si face of the flavin
inaccessible to solvent.
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Figure 3. Active site of the DrPRODHo−THFA complex (relaxed stereographic views). (A) Electron density map for the DrPRODHo−THFA
complex. The cage represents a simulated annealing σA-weighted Fo − Fc omit map (3.0σ). (B) Superposition of the DrPRODHo−THFA complex
(yellow) and the PutA86−669−THFA complex (gray, PDB entry 1tiw), highlighting differences in the orientation of the conserved glutamate of α8
(Glu295 in DrPRODH, Glu559 in PutA). Yellow and black dashes denote the unique electrostatic interactions of DrPRODH and PutA86−669,
respectively. Conformations A and B of the FAD in the DrPRODHo−THFA complex are colored yellow and pink, respectively.
with the conserved Glu559 of α8 (Figure 3B), which
corresponds to Glu295 of DrPRODH. Glu295, however,
adopts a different conformation and is thus unable to form
an analogous hydrogen bond with the 4′-OH (Figure 3B).
Ribityl B is more familiar. It is similar to the ribityl of ligand-
free TtPRODH (Figure 5B). In both structures, the 2′- and 3′-
hydroxyls are below the pyrimidine ring, while the 4′-OH is
below the dimethylbenzene ring. One notable difference is that
the 4′-OH of DrPRODH ribityl B is located on the si face of
the FAD, whereas that of TtPRODH is torsioned over to the re
side. The re side location allows the hydroxyl to engage a
structurally conserved water molecule that bridges the main
chains of β5 and β6 in all PRODH and PutA structures (Figure
5B).
The flavins of DrPRODH and PutA also differ in the position
of the adenosine group (Figure 5A). Because of a rotation
around the pyrophosphate, the adenine ring positions of the
two structures differ by 13 Å. This difference was also noted in
TtPRODH and is caused by the presence of an extra α-helix in
PutAs that is absent in monofunctional PRODHs.21
which are observed in both molecules in the asymmetric unit.
Large protein conformational changes are also observed in
chain A of the asymmetric unit, and the electron density map is
consistent with release of THFA. In the other chain, crystal
contacts prevent large protein conformational changes, and the
electron density map suggests that an acetate ion replaces
THFA. We note that PutA86−669 crystallized in PEG 3350
also has an acetate ion bound in the active site.28 The
discussion below focuses on chain A, because it provides
information about conformational changes in both the flavin
and the protein.
Reduction by dithionite induces substantial conformational
changes in the flavin. The reduced flavin exhibits a 20° butterfly
bend in the isoalloxazine (Figure 4C), which contrasts the
planar isoalloxazine of the oxidized enzyme. Severe butterfly
bending (20−30°) has also been observed in the dithionite-
reduced flavin of PutA86−66928 and the covalently modified,
reduced flavins of N-propargylglycine-inactivated PRODH29
and PutA.30 The bending of the flavin pushes the N5−N10 axis
toward the proline binding site, which results in the si face
being convex. The reduced flavin of DrPRODHr exhibits just
one ribityl conformation (Figure 4B), which is nearly identical
to the ribityl chains of the oxidized FAD in TtPRODH and the
Structure of DrPRODHr. A 1.75 Å resolution structure of
DrPRODHr was determined from a crystal of the DrPROD-
Ho−THFA complex that was soaked in sodium dithionite.
Reduction causes large conformational changes in the flavin,
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Figure 4. Conformations of the flavin in DrPRODH (relaxed stereographic views). (A) Dual FAD conformations of the DrPRODHo−THFA
complex. Conformations A (q = 0.56) and B (q = 0.44) are colored yellow and pink, respectively. Yellow and red dashes denote the unique hydrogen
bonds of conformations A and B, respectively. Note that the two conformations differ mainly in the orientations of the 2′-OH and 3′-OH groups of
the ribityl. (B and C) Two views of the dithionite-reduced flavin of DrPRODHr. In all three panels, the cage represents a simulated annealing σA-
weighted Fo − Fc omit map (3.0σ).
reduced flavin in the N-propargylglycine-inactivated E. coli PutA
PRODH domain30 (Figure 5C).
we note that electron density for the β1−α1 loop is relatively
weak in DrPRODHr, implying high mobility. In fact, density for
the side chain of Glu64 in DrPRODHr is absent, indicating that
the side chain is disordered. Electron density for the side chain
of Arg291 is likewise weak but suggests that the guanidinium
group turns away from the proline binding site (Figure 6A).
Flavin reduction also induces disorder in another key side chain
of α8, Glu295, which hydrogen bonds with the FAD adenine
and ribityl groups in the THFA complex.
The large movements of α8 and the β1−α1 loop dismantle
the proline binding site and profoundly change the solvent
accessibility of the flavin. In the THFA complex, the
isoalloxazine and THFA are buried beneath the Arg291−
Glu64 ion pair (Figure 6B), and the interaction between
Flavin reduction also dramatically changes the protein
conformation. Upon flavin reduction, α8 shifts away from the
isoalloxazine by 1.7 Å, and the β1−α1 loop (residues 62−69)
withdraws from the active site by >5 Å (Figure 6A). One major
consequence of these movements is the rupture of the Arg291−
Glu64 ion pair, which links α8 and the β1−α1 loop in the
THFA complex. The electron density for the ion pair in the
THFA complex is exceptional, indicating that this interaction is
quite strong (Figure 3A). Its rupture is thus a significant event.
Breaking of the ion pair causes Glu64 and Arg291 to separate
from each other. Glu64 is driven to the protein surface along
with the rest of the β1−α1 loop (Figures 2B and 6A), although
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Figure 6. Conformational changes induced by flavin reduction and
product release. (A) Superposition of the active sites of the
DrPRODHo−THFA complex (yellow) and DrPRODHr (gray)
highlighting the shift of α8, reconfiguration of the β1−α1 loop, and
breaking of the Arg291−Glu64 ion pair. Black dashes indicate
electrostatic interactions in the DrPRODHo−THFA complex. (B)
Surface representation of the DrPRODHo−THFA complex. FAD and
THFA are drawn as yellow and pink spheres, respectively. Glu64 and
Arg291 are colored red and blue, respectively. Glu295 is colored green.
The inset shows a view from the bottom of the active site. (C) Surface
representation of DrPRODHr. The coloring is the same as in panel B.
The dashes outline the openings to the active site.
Figure 5. Comparison of flavin conformations in PRODHs and PutAs.
(A) Superposition of FAD conformation A of the DrPRODHo−THFA
complex (yellow) and the FAD of the PutA86−669−THFA complex
(salmon, PDB entry 1tiw). (B) Superposition of FAD conformation B
of the DrPRODHo−THFA complex (pink) and the oxidized flavin of
TtPRODH (cyan, PDB entry 2g37). (C) Superposition of the reduced
flavin of DrPRODHr (gray), the oxidized FAD of TtPRODH (cyan),
and the inactivated flavin of the PutA PRODH domain (magenta, PDB
entry 3ITG).
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Table 2. Kinetic Parameters for DrPRODH and DrPRODH Mutant Enzymes Using Proline and CoQ1 as the Substrates
kcat (s−1
)
Km (mM)
kcat/Km (s−1 M−1
)
variational effect (kcat/Km of mutant)/(kcat/Km of DrPRODH)
a
Proline as the Variable Substrate
30 4.5
DrPRODH
G63A
8.7 0.58
0.080 0.006
0.055 0.001
290 39
384 68
0.21 0.04
0.007 0.002
0.037 0.007
E64A
50
6
1.1 0.13
b
CoQ1 as the Variable Substrate
90323 24185
DrPRODH
G63A
14
1
0.155 0.04
0.028 0.01
0.043 0.003
0.046 0.003
1535 559
0.017 0.008
0.016 0.007
E64A
0.032 0.01
1438 458
a
b
The CoQ1 concentration was fixed at 200 μM. The proline concentration was fixed at 500 mM.
Glu295 and the ribityl seals the bottom of the active site
(Figure 6B, inset). In the reduced enzyme, the separation of
Arg291 from Glu64 creates a large, solvent-exposed cavity in
the upper part of the active site, which contains the
isoalloxazine (Figure 6C). Furthermore, the combination of
disorder in Glu295 and the change in the ribityl conformation
creates a hole in the bottom of the active site of the reduced
enzyme (Figure 6C, inset). Thus, the active site of the reduced,
ligand-free enzyme is open, and the flavin is highly solvent
exposed.
that of DrPRODH. In summary, the kinetic analysis of G63A
and E64A confirms the significance of the conserved sequence
motif and is consistent with the structures, which suggest that
Gly63 is important for the flexibility of the β1−α1 loop and
Glu64 is important for stabilizing the closed active site.
DISCUSSION
■
The DrPRODH structures provide new information about the
conformations populated by monofunctional PRODHs during
the reductive half-reaction (Figure 1B). In particular, the
DrPRODHo−THFA structure represents the E−S complex
[state II (Figure 1B)]. The active site of the E−S complex is
closed, and the substrate is buried. The tight packing within the
complex is also observed in PutA86−669 (Figure 3B) and
presumably helps align proline with the flavin for the hydride
transfer. A water molecule is present in the E−S complex,
which bridges conserved Tyr193 and the THFA heteroatom (N
atom of proline). An analogous water molecule is also present
in the PutA86−669−THFA complex (Figure 3B), suggesting
that it is conserved in the PRODH superfamily. It is tempting
to speculate that this water molecule hydrolyzes P5C to GSA. If
so, the hydrolysis reaction occurs within the active site rather
than in solution as implied in Figure 1A and most biochemistry
textbooks and literature articles on proline catabolism.
Kinetic Parameters of DrPRODH and DrPRODH β1−α1
Loop Mutants. The observation of large conformational
changes in the β1−α1 loop induced by flavin reduction and
release of THFA prompted an investigation of this loop using
site-directed mutagenesis. As described by White et al., the
β1−α1 loop harbors one of nine conserved sequence motifs
that define the PRODH superfamily.21 This motif includes a
Gly-Glu pair that is present in all monofunctional PRODHs
and PutAs and corresponds to the Gly63-Glu64 pair of
DrPRODH. These residues were therefore investigated by
creating the site-directed mutant enzymes G63A and E64A.
The kinetic parameters for wild-type DrPRODH were first
determined (Table 2). The values of kcat and Km using proline
as the variable substrate with a fixed CoQ1 concentration are
8.7 s−1 and 290 mM, respectively, resulting in a kcat/Km of 30
s−1 M−1. For comparison, the corresponding parameters of the
closely related TtPRODH (47% identical to DrPRODH) are as
follows: kcat = 13 s−1, and Km = 27 mM.21 Those of the E. coli
PutA are as follows: kcat = 5.2 s−1, and Km = 42 mM.27 Thus, the
Km for proline of DrPRODH is higher than expected. The
kinetic parameters were also determined using CoQ1 as the
variable substrate at fixed proline concentration. These values
are as follows: kcat = 14 s−1, and Km = 155 μM. The
corresponding values for TtPRODH are not available. Those of
the E. coli PutA are as follows: kcat = 3.4 s−1, and Km = 110
μM.27 Finally, THFA was found to inhibit DrPRODH
competitively with proline. The estimated KI value of 38 mM
is >10 times higher than those of TtPRODH (1 mM21) and
PutA (1.6 mM27). These results suggest that the affinity of
DrPRODH for proline is atypically low.
The E−S complexes of DrPRODH and PutA86−669 are
very similar (Figure 3B). In both cases, the inhibitor is
completely buried between the si face of the FAD and α8. The
enzyme−inhibitor interactions and ion pair gate conformation
are virtually identical in the two structures. Thus, substrate
recognition is highly conserved in the PRODH superfamily.
The only notable difference involves the conserved glutamate
of α8 (Glu295 of DrPRODH and Glu559 of PutA). The
different orientations of this side chain seem to be related to the
different conformations of the FAD adenosine groups. In
DrPRODH, the adenine ring packs against α8 and forms a
hydrogen bond with Glu295 (Figure 3B). This interaction is
not possible in PutA because of the markedly different location
of the adenosine (Figure 5A), which is caused by the presence
of an extra α-helix in PutAs that is absent in monofunctional
PRODHs.21
Catalytic activity is severely impaired in mutants G63A and
E64A (Table 2). Using proline as the varying substrate, kcat
values of 0.08 and 0.055 s−1 were obtained for G63A and E64A,
respectively, which are >100 times lower than that of
DrPRODH. The catalytic efficiencies of G63A and E64A for
proline are 140- and 27-fold lower than that of DrPRODH,
respectively. Using CoQ1 as the varying substrate, the kcat
values are 0.043 and 0.046 s−1 for G63A and E64A, respectively,
which are 300-fold lower than that of DrPRODH. The
efficiencies of the mutants for CoQ1 are 60-fold lower than
Given the sequence and structural conservation of the
proline binding site, it is surprising that DrPRODH has a
markedly lower substrate affinity than PutA and TtPRODH.
One possibility is that the FAD ribityl plays a role in tuning
substrate affinity. DrPRODH is unique in that two con-
formations of the ribityl are observed in the E−S complex,
neither of which corresponds to that of the high-affinity
PutA86−669−THFA complex, although conformation A is
reminiscent of the flavin in the PutA86−669−THFA complex.
It is possible that conformation B represents an inactive state
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and that a conformational change is required to transform it to
conformation A before hydride transfer occurs. If so, Glu295
may play a role in this transformation because it hydrogen
bonds with the 3′-OH of conformation B (Figure 4A).
Additional structures of PRODH−THFA complexes would
shed light on this issue.
elements in PutA that are absent in monofunctional PRODHs,
rupture of the ion pair and shifting of α8 in trifunctional PutA
could help initiate a cascade of events that eventually leads to a
larger, more global isomerization of the protein that unveils a
high-affinity membrane association domain.
Finally, although it was not possible to crystallize DrPRODH
in the absence of THFA, presumably the active site of the
oxidized enzyme is open prior to substrate binding (state I in
Figure 1B). Indeed, this is true for TtPRODH. The structure of
oxidized, ligand-free TtPRODH resembles DrPRODHr in that
the ion pair gate is open and α8 is shifted away from the
isoalloxazine (Figure 2B). In fact, the shift of α8 in TtPRODH
is 1.3 Å larger than that of DrPRODHr (Figure 2B). Whether
substrate binding to the open enzyme induces closure of the
gate (induced fit) or the substrate binds to closedlike
conformations that arise from thermal fluctuations (conforma-
tional selection) remains to be determined. However, the large
conformational changes that are required to form the closed E−
S complex seem to favor conformational selection as a plausible
mechanism for substrate binding.33
Comparison of the DrPRODHo−THFA and DrPRODHr
structures provides insight into conformational changes that
occur upon flavin reduction and product release (state II to
state IV in Figure 1B). These include severe bending of the
isoalloxazine, dihedral rotation of the ribityl chain, shifting of
α8, reconfiguration of the β1−α1 loop, rupture of the
conserved Arg291−Glu64 ion pair, and disordering of Glu295.
The structures suggest a scenario for how the enzyme moves
from state II to state IV of the reductive half-reaction. The
butterfly bending of the isoalloxazine appears to initiate a
cascade of events that leads to product release. Bending of the
ring system pushes the N5−N10 axis of the flavin toward the
newly formed product, creating steric clash in the highly
crowded active site. The shifting of α8 and rupture of the
Arg291−Glu64 ion pair alleviate this clash, creating a large
cavity that presumably allows facile product dissociation.
The Arg291−Glu64 ion pair is a central player in the
conformational changes observed in DrPRODH. Because it is
formed in the closed E−S complex and broken in the ligand-
free state, the ion pair appears to function as the active site gate.
Because the ion pairing residues are identically conserved in
monofunctional PRODHs and PutAs, we suggest that the gate
is a universal aspect of PRODH catalysis.
ASSOCIATED CONTENT
■
S
* Supporting Information
Electron density evidence for dual FAD conformations in the
DrPRODHo−THFA complex (Figure S1). This material is
available free of charge via the Internet at http://pubs.acs.org.
Accession Codes
Atomic coordinates and structure factors have been deposited
in the Protein Data Bank as entries 4H6Q (DrPRODHo−
THFA) and 4H6R (DrPRODHr).
Whereas the ion pair gate opens to the bulk medium in
DrPRODH, the gate of PutA separates the proline binding site
from a large, internal cavity that traverses 41 Å to the P5CDH
active site.20 This cavity serves as a conduit through which
P5C/GSA is channeled to the P5CDH active site. In the
structure of full-length B. japonicum PutA, the gate is closed and
a sulfate ion is bound in the PRODH active site. A structure of
this enzyme with the proline site empty is not available, but on
the basis of the DrPRODH structures, we predict that
reduction of the FAD in PutA opens the gate, allowing P5C/
GSA to enter the substrate channeling cavity. Curiously,
dithionite reduction of the crystalline PutA86−669−THFA
complex does not break the ion pair, although the flavin
conformation is altered as in DrPRODHr.28 It is possible that
crystal contacts inhibit rupture of the gate in crystalline
PutA86−669. Clearly, new structures of full-length PutAs are
needed to understand how the ion pair gate functions in PutA.
The structural changes observed in DrPRODH perhaps also
provide insight into functional switching of trifunctional PutAs.
Trifunctional PutA is a type of flavin switch protein that serves
as a transcriptional repressor of the put regulon in addition to
having PRODH and P5CDH catalytic activities.31 How
trifunctional PutA switches from being a DNA-bound tran-
scriptional repressor to membrane-associated bifunctional
enzyme (also known as functional switching) is a major
question in PutA biochemistry. Recent rapid reaction kinetic
measurements of the paradigmatic trifunctional PutA from E.
coli revealed an isomerization step that occurs after reduction of
FAD, and this step was proposed to report on flavin-dependent,
global conformational changes that drive functional switching.32
Because rupture of the ion pair gate and movement of α8 occur
after flavin reduction in DrPRODH, it is possible that
analogous changes are also part of functional switching.
Because α8 and the β1−α1 loop contact other structural
AUTHOR INFORMATION
■
Corresponding Author
*Department of Chemistry, University of MissouriColumbia,
Columbia, MO 65211. Phone: (573) 884-1280. Fax: (573)
882-2754. E-mail: tannerjj@missouri.edu.
Funding
Research reported in this publication was supported by the
National Institute of General Medical Sciences of the National
Institutes of Health via Grant GM065546.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank Drs. Jonathan Schuermann and Jay Nix for help with
X-ray data collection and processing. We thank Dr. Ray Stevens
and the Joint Center for Structural Genomics for providing the
DrPRODH clone and Dr. Christopher Hill for providing the
pKA8H plasmid. 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 DE-AC02-05CH11231. This work is based upon
research conducted at the Advanced Photon Source on the
Northeastern Collaborative Access Team beamlines, which are
supported by Grant RR-15301 from the National Center for
Research Resources at the National Institutes of Health. Use of
the Advanced Photon Source, an Office of Science User Facility
operated for the U.S. Department of Energy (DOE) Office of
Science by Argonne National Laboratory, was supported by the
U.S. DOE under Contract DE-AC02-06CH11357.
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ABBREVIATIONS
■
PRODH, proline dehydrogenase; P5C, 1-pyrroline-5-carbox-
ylate; GSA, glutamate semialdehyde; P5CDH, 1-pyrroline-5-
carboxylate dehydrogenase; POX, human proline oxidase;
TtPRODH, proline dehydrogenase from T. thermophilus;
PutA, proline utilization A; DrPRODH, proline dehydrogenase
from D. radiodurans R1; DrPRODHo, oxidized proline
dehydrogenase from D. radiodurans R1; DrPRODHr, reduced
proline dehydrogenase from D. radiodurans R1; THFA, L-
tetrahydrofuroic acid; CoQ1, coenzyme Q1; TEVP, tobacco
etch virus protease; PutA86−669, PRODH construct of E. coli
PutA containing residues 86−669.
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