Structural Analysis of Prolines and Hydroxyprolines Binding to the L-glutamate-γ-semialdehyde Dehydrogenase Active Site of Bifunctional Proline Utilization A

Abstract

Proline utilization A (PutA) proteins are bifunctional proline catabolic enzymes that catalyze the 4-electron oxidation of L-proline to L-glutamate using spatially-separated proline dehydrogenase and L-glutamate-γ-semialdehyde dehydrogenase (GSALDH, a.k.a. ALDH4A1) active sites. The observation that L-proline inhibits both the GSALDH activity of PutA and monofunctional GSALDHs motivated us to study the inhibition of PutA by proline stereoisomers and analogs. Here we report five high-resolution crystal structures of PutA with the following ligands bound in the GSALDH active site: D-proline, trans-4-hydroxy-D-proline, cis-4-hydroxy-D-proline, L-proline, and trans-4-hydroxy-L-proline. Three of the structures are of ternary complexes of the enzyme with an inhibitor and either NAD⁺ or NADH. To our knowledge, the NADH complex is the first for any GSALDH. The structures reveal a conserved mode of recognition of the inhibitor carboxylate, which results in the pyrrolidine rings of the D- and L-isomers having different orientations and different hydrogen bonding environments. Activity assays show that the compounds are weak inhibitors with millimolar inhibition constants. Curiously, although the inhibitors occupy the aldehyde binding site, kinetic measurements show the inhibition is uncompetitive. Uncompetitive inhibition may involve proline binding to a remote site or to the enzyme-NADH complex. Together, the structural and kinetic data expand our understanding of how proline-like molecules interact with GSALDH, reveal insight into the relationship between stereochemistry and inhibitor affinity, and demonstrate the pitfalls of inferring the mechanism of inhibition from crystal structures alone.

Graphical Abstract

1. Introduction

The enzymes of proline catabolism catalyze the 4-electron oxidation of L-proline to L-glutamate (Scheme 1) [1, 2]. The first step is the oxidization of L-proline to Δ¹-pyrroline-5-carboxylate (P5C), catalyzed by FAD-dependent proline dehydrogenase (PRODH). P5C forms a pH-dependent equilibrium with its hydrolysis product L-glutamate-γ-semialdehyde (GSAL). The latter is the substrate for the last enzyme of proline catabolism, GSAL dehydrogenase (GSALDH, a.k.a. ALDH4A1), which catalyzes the NAD⁺-dependent oxidation GSAL to L-glutamate. The enzymes PRODH and GSALDH are widely conserved in eukaryotes and bacteria. In some bacteria, PRODH and GSALDH are combined into the bifunctional enzyme known as proline utilization A (PutA) [3].

Scheme 1.

Reactions of proline catabolism.

PutAs are large enzymes (>1000 residues) that catalyze the oxidation of L-proline to L-glutamate using PRODH and GSALDH active sites. The crystal structures of PutAs have revealed a conserved fold in which the two active sites are separated by ~40 Å and connected by a narrow tunnel [4–8]. The tunnel implies a substrate channeling mechanism, which has been confirmed in several PutAs [5, 6, 9–11].

Substrate inhibition is an interesting aspect of PutAs. The coupled PRODH-GSALDH activity of PutA is inhibited by L-proline, the substrate of the PRODH active site. Substrate inhibition has been observed with several PutAs, with the K_i values in the range of range of 24 – 263 mM [6, 9, 10]. Substrate inhibition likely has physiological relevance, given that the K_m for proline is 7 – 56 mM [6, 9, 10]. A recent crystal structure of PutA showed that the basis of substrate inhibition is the binding of L-proline in the GSALDH site [12]. This observation is consistent with other studies showing that L-proline is an inhibitor of human GSALDH, which is a monofunctional enzyme [13]. It has been suggested that the inhibition of PutA by proline may be advantageous during osmotic stress, when bacteria need to accumulate high levels of proline rather than catabolizing it [12].

The inhibition of PutA and monofunctional GSALDH by L-proline motivated us to ask a wider molecular recognition question of whether the GSALDH active site can accommodate proline stereoisomers and analogs. Here we report the high-resolution crystal structures of PutA from Sinorhizobium meliloti (SmPutA) with five different proline molecules bound in the GSALDH active site (Scheme 2): L-proline, trans-4-hydroxy-L-proline (THLP), D-proline, cis-4-hydroxy-D-proline (CHDP), and trans-4-hydroxy-D-proline (THDP). The structures reveal a conserved mode of recognition of the carboxylate group, which results in the pyrrolidine rings of the D- and L-isomers having different orientations and different hydrogen bonding environments. Kinetic measurements suggest that hydroxyprolines are weak inhibitors of the SmPutA GSALDH domain. The structures provide insight into the recognition of proline-like compounds by GSALDH, which could be useful for developing hydroxyproline-based inhibitors of proline metabolism.

Scheme 2.

Chemical structures of proline and hydroxyproline compounds used in this study.

2. Materials and methods

2.1. Materials

The following compounds were purchased from Sigma: L-proline (product number P0380), D-proline (product number 858919), trans-4-hydroxy-L-proline (THLP, product number H54409), trans-4-hydroxy-D-proline (THDP, product number 702501), cis-4-hydroxy-D-proline (CHDP, product number H5877), cis-4-hydroxy-L-proline (CHLP, product number H1637).

2.2. Crystallization

SmPutA was expressed in Escherichia coli and purified as described previously [14]. SmPutA was co-crystallized with ligands at 13 °C using the sitting-drop vapor diffusion method. Crystallization experiments were set up with SmPutA (6 mg/mL) in a buffer containing 50 mM Tris (pH 8.0), 50 mM NaCl, 5% (w/v) glycerol, and 0.5 mM Tris(2-caboxyethyl)phosphine. Crystals were grown using a reservoir solution containing 19% PEG-3350, 0.2 M ammonium sulfate, 0.1 M magnesium chloride, 0.1 M HEPES (pH 8.0), and 0.1 M sodium formate, and the drops were formed by combining 2 µL of the reservoir and 2 µL of the protein-ligand solution.

Crystals of SmPutA complexed with CHDP or THLP were produced by co-crystallization with 50 mM of the proline ligand and 10 mM NAD⁺. Crystals of SmPutA complexed THDP or D-proline were produced by co-crystallization with 50 mM of the proline ligand without NAD⁺. We note that similar co-crystallization experiments were set up with CHLP, but no ligand density was observed in the active site, so the structure is not reported. All crystals were cryoprotected in reservoir buffer with 15 % additional PEG-200, and flash cooled.

A different approach was used to capture the complex with L-proline in crystallo. First, crystals of SmPutA complexed with NADH were produced by co-crystallization with 10 mM NADH. Then, the crystals were soaked briefly (~1 min) with 50 mM L-proline during cryoprotection. Short soaking with L-proline was done to minimize the generation of P5C by the PRODH active site, which might have complicated the interpretation of electron density in the GSALDH site. NADH was included rather than NAD⁺ to avoid turnover by the GSALDH site. The high concentration of L-proline used ensured that any P5C/GSA generated was unlikely to bind to the GSALDH active site.

2.3. X-ray diffraction data collection and refinement

X-ray diffraction data were collected in shutterless mode at beamline 4.2.2 of the Advanced Light Source (Taurus-1 CMOS detector). The data were integrated and scaled using XDS [15]. Intensities were converted to amplitudes using AIMLESS [16]. All the data sets are in space group P2₁ and have similar unit cell dimensions of a = 101 – 102 Å, b = 102 – 103 Å, c = 126 – 127 Å, and β =106°. The asymmetric unit contains a dimer of SmPutA. We note this is the same crystal form used for previous crystallographic studies of SmPutA [6, 14]. Data processing statistics are listed in Table 1.

Table 1.

Data Collection and Refinement Statistics

	D-Proline	THDP	CHDP NAD+	L-Proline NADH	THLP NAD+
Space group	P21	P21	P21	P21	P21
Unit cell	a = 101.21	a = 101.15	a = 100.89	a = 101.08	a = 101.32
parameters (Å,°)	b = 102.32	b = 102.00	b = 101.92	b = 102.25	b = 102.14
	c = 127.03	c = 126.75	c = 126.20	c = 127.12	c = 126.46
	β = 106.42	β = 106.45	β = 106.44	β = 106.48	β = 106.42
Wavelength (Å)	0.9762	1.0000	1.0000	1.0000	1.0000
Resolution (Å)	46.08 – 1.56	47.03 – 1.41	48.38 – 1.46	48.46 – 1.44	48.59 – 1.54
	(1.59 – 1.56)	(1.43 – 1.41)	(1.48 – 1.46)	(1.46 – 1.44)	(1.57 – 1.54)
Observationsa	1255558 (60757)	1532777 (41774)	1480175 (25200)	1484873 (44356)	1673607 (63243)
Unique reflectionsa	350164 (17275)	456940 (18972)	385095 (11043)	412199 (13329)	363339 (17948)
Rmerge(I)a	0.082 (1.272)	0.060 (1.322)	0.062 (1.298)	0.059 (1.263)	0.087 (1.297)
Rmeas(I)a	0.097 (1.504)	0.071 (1.752)	0.071 (1.684)	0.069 (1.506)	0.098 (1.535)
Rpim(I)a	0.050 (0.795)	0.038 (1.133)	0.033 (1.055)	0.036 (0.810)	0.043 (0.813)
Mean I/σa	8.1 (0.6)	10.7 (0.5)	12.2 (0.5)	10.9 (0.8)	11.2 (0.9)
CC1/2	0.998 (0.374)	0.998 (0.259)	0.998 (0.323)	0.999 (0.365)	0.998 (0.378)
Completeness (%)a	95.5 (99.0)	96.5 (81.3)	91.0 (52.8)	92.4 (60.4)	99.8 (99.8)
Multiplicitya	3.6 (3.5)	3.4 (2.2)	3.8 (2.3)	3.6 (3.3)	4.6 (3.5)
No. of protein residues	2430	2427	2428	2417	2435
No. of atoms
Protein	18154	18104	18006	17989	18134
FAD	106	106	106	106	106
Pro ligand	24	27	18	24	18
NAD(H)	N/A	N/A	88	132	88
Water	2395	2521	1998	1992	2211
Rcryst	0.1742 (0.3226)	0.1752 (0.3702)	0.1937 (0.3560)	0.1798 (0.3240)	0.1792 (0.2997)
Rfreeb	0.2037 (0.3318)	0.1988 (0.3713)	0.2200 (0.3890)	0.2053 (0.3459)	0.2104 (0.3308)
rmsd bonds (Å)	0.005	0.005	0.005	0.005	0.006
rmsd angles (°)	0.820	0.823	0.844	0.838	0.839
Ramachandran plotc
Favored (%)	98.47	98.47	98.22	98.29	98.35
Outliers (%)	0.00	0.04	0.00	0.04	0.00
Clashscore (PR)c	1.61 (99)	1.69 (99)	2.06 (99)	2.00 (99)	1.85 (99)
MolProbity score (PR)c	0.91 (100)	0.92 (100)	0.98 (100)	0.97 (99)	0.95 (100)
Average B (Å2)
Protein	21.5	21.0	22.8	25.1	20.9
FAD	18.6	20.6	21.7	20.2	18.8
Pro ligand	21.0	24.2	20.0	31.3	21.9
NAD(H)	N/A	N/A	19.0	19.4	17.9
Water	29.3	30.5	30.0	31.6	28.6
Coord. error (Å)d	0.20	0.18	0.19	0.17	0.20
PDB code	6X99	6X9A	6X9B	6X9C	6X9D

^aValues for the outer resolution shell of data are given in parenthesis.

^b5% test set.

^cFrom MolProbity. The percentile ranks (PR) for Clashscore and MolProbity score are given in parentheses.

^dMaximum likelihood-based coordinate error estimate from PHENIX.

PHENIX [17] was used for refinement and Coot [18] was used for model building. The starting model for refinement was derived from a previous structure of SmPutA (PDB ID: 5KF6). SMILES strings for L-proline, D-proline, THDP, CHDP, and THLP, were used as the input to ELBOW [19] to generate the ligand coordinates and restraint files used during refinement. Structure validation was performed using MolProbity and the wwPDB validation service [20, 21]. Ligand modeling was validated using polder omit maps [22]. Refinement statistics can be found in Table 1.

2.4. Fluorescence-based thermal shift assays (TSA)

SmPutA was diluted to 1 mg/mL in a buffer containing 50 mM Tris (pH 8.0), 50 mM NaCl, 5% (w/v) glycerol, and 0.5 mM Tris(2-caboxyethyl)phosphine. CHLP, CHDP, THLP or THDP was added to achieve a final concentration of 125 mM. The enzyme-ligand solution was mixed 1:1 with a 2 × SYPRO orange solution (Thermo Fisher Scientific), and the samples were incubated for 45 minutes in the dark at 4 °C to allow time to equilibrate with SYPRO orange. The total volume of each sample was 20 µL. The stability curves of the enzyme-ligand complexes were measured in a QuantStudio version 3 real-time PCR system (Thermo Fisher Scientific) using a MicroAmp 96-well optical plate (Thermo Fisher Scientific). The temperature was raised from 4 °C to 95 °C in 0.5 °C steps with a 20-s hold following each step while the fluorescence was measured. Stability curves were analyzed using the server https://beamerlab.shinyapps.io/software/ and T_1/2 was determined as previously described [23].

2.5. GSALDH activity measurements

The GSALDH activity of SmPutA in the presence of inhibitors was measured by monitoring NADH production at 340 nm with L-P5C as the variable substrate (0 – 1.25 mM) and NAD⁺ as the fixed substrate (0.2 mM). D,L-P5C was synthesized from D,L-5-hydroxylysine-HCl according to the method of Williams and Frank [24]. The concentrations of D,L-P5C was determined by adding ortho-aminobenzaldehyde (o-AB) and monitoring the absorbance at 443 nm, assuming an extinction coefficient for the P5C-o-AB adduct of 2.59 mM⁻¹cm⁻¹. For use in kinetics, D,L-P5C was neutralized to pH 7.5 immediately prior to assays using 1 M Tris (pH 7.5) and 6 M NaOH. The concentration of L-P5C was assumed to be half the total D,L-P5C concentration added to the assays. The data were acquired at room temperature in 96-well plates using a BioTek Epoch 2 microplate spectrophotometer. The assay buffer contained 100 mM sodium phosphate pH 7 and 1 mM EDTA. The SmPutA concentration in each assay was 170 nM. The initial rates were estimated from linear regression of the first 5 – 6 minutes of the progress curve. The initial rate data for each inhibitor were fit globally with Origin software to various inhibition models, including competitive, uncompetitive, mixed, and competitive with substrate inhibition [25].

3. Results

3.1. Electron density for ligands bound in the GSALDH active site of SmPutA

A bifunctional PRODH-GSALDH enzyme (SmPutA) was used to determine high-resolution structures of a GSALDH active site complexed with prolines and hydroxyprolines. The PRODH and GSALDH active sites in SmPutA are separated by ~40 Å (Fig. 1A). Co-crystallization and crystal soaking experiments were performed with the compounds L-proline, D-proline, CHLP, THLP, CHDP and CHLP (Scheme 2). For all the compounds except CHLP, electron density was observed in the GSALDH active site and the compounds could be built into the density with occupancy of 1.0 (Fig. 1B). Electron density for CHLP was not evident, suggesting it does not bind GSALDH at the concentration used (50 mM). No electron density for any of these ligands was observed in the PRODH site.

Fig. 1.

Structure of SmPutA and electron density evidence for prolines bound in the GSALDH active site. (A) Cartoon representation of SmPutA complexed with THDP (cyan). The protein is colored in a rainbow scheme with blue at the N-terminus and red at the C-terminus. (B) Polder omit electron density for the bound ligands (contoured at 4σ).

Three of the structures are of ternary complexes, which include the cofactor NAD(H) bound to the GSALDH active site. Strong electron density for NAD⁺ was observed in the CHDP and THLP complex structures (Figs. 2A and 2B). NAD⁺ has an extended conformation in these structures, with the oxidized nicotinamide ring near the catalytic cysteine residue and positioned to accept a hydride ion from the hemithioacetal intermediate. This is the canonical conformation of NAD⁺ bound to Rossmann fold proteins and is known as the “hydride transfer conformation”. In contrast, the L-proline complex was determined from a crystal of SmPutA co-crystallized with NADH. NADH adopts a more compact conformation in which the reduced nicotinamide has retracted from the active site (Fig. 2C). This conformation is known as the “hydrolysis conformation” [26]. Recoil of the cofactor following hydride transfer allows room for a water molecule to enter the active site for hydrolysis of the acyl-enzyme intermediate. Curiously, in chain B of the L-proline complex, the density is consistent with NADH adopting both the hydrolysis and hydride transfer conformations with occupancies of 0.56 and 0.44, respectively (Fig. 2D). To our knowledge, this is the first structure of a GSALDH complexed with NADH.

Fig. 2.

Electron density for NAD(H) bound to SmPutA. (A) NAD⁺ in the CHDP complex, exhibiting the hydride transfer conformation. (B) NAD⁺ in the THLP complex, exhibiting the hydride transfer conformation. (C) NADH in the hydrolysis conformation observed in chain A of the L-proline complex. (D) Dual conformations of NADH in chain B of the L-proline complex. The hydrolysis conformation is colored salmon; the hydride transfer conformation is colored gray.

3.2. Poses and interactions of D,L-prolines and D,L-4-hydroxyprolines bound to GSALDH

The five proline compounds exhibit the pose expected for a carboxylate compound bound to GSALDH. The carboxylate group is directed toward the anchor loop (residues 1001–1003), and the pyrrolidine ring is wedged between Phe708 and Phe1010 (Fig. 3). This general binding mode is consistent with other GSALDH structures, where the carboxylate interacts with the anchor loop and the nonpolar parts of the ligand are flanked by the two phenylalanine rings [27, 28]. We note that Phe708 and Phe1010 form the so-called “aromatic box”, a conserved feature of substrate recognition by ALDH superfamily enzymes [27, 29, 30]. The protein conformations are nearly identical in the five complexes, except for variations in the side chains of Phe708 and Glu674 (Fig. 3).

Fig. 3.

Superposition of the five structures emphasizing the orientations of the inhibitors relative to catalytic Cys844, the aromatic box, and Glu674.

The complexes exhibit a conserved set of interactions with the carboxylate group. In each structure, the carboxylate forms an ion pair with Arg483, as well as hydrogen bonds with Gly1002, Ala1003, and Ser845 (Fig. 4). In addition, the carboxylate hydrogen bonds with a conserved water molecule (“cw” in Fig. 4).

Fig. 4.

Electrostatic interactions for D,L-prolines bound to GSALDH: (A) D-proline, (B) THDP, (C) CHDP, (D) L-proline, and (E) THLP. The dashes represent hydrogen bonds and ion pairs calculated with a cutoff distance of 3.2 Å. “cw” denotes conserved water. Donor-acceptor distances (in Å) are shown for interactions involving Glu674; the value listed is the average for the two chains in the asymmetric unit.

The conserved pose of the carboxylate results in the pyrrolidine rings of the D- and L-isomers having different orientations. In the D-isomers, the pyrrolidine ring is approximately perpendicular to the phenyl rings of the aromatic box, and the amino group sits on the Phe708 side of the aromatic box (Fig. 3). In the L-isomers, the pyrrolidine ring is roughly parallel to the aromatic box residues, with the amino group pointing toward Glu674 (Fig. 3).

The difference in the pyrrolidine ring orientation has two main consequences. First, an additional water molecule is found in the active sites of the D-isomer complexes (Figs. 4A–4C). This water molecule bridges the carboxylate of the inhibitor with the backbone amino groups of Cys844 and Ser845. In the L-isomer complexes, the C3 of the inhibitor blocks this solvent site. Second, the amino groups of the L-isomers are able to engage the carbonyl of Ala1003 either by a direct hydrogen bond (THLP) or a water-mediated one (L-Pro) (Figs. 4D and 4E).

3.3. Recognition of the 4-hydroxyl group of D,L-hydroxyproline

The structures of SmPutA complexed with three different 4-hydroxyprolines were determined: THDP, CHDP, and THLP. Regardless of the stereochemistry at C2, the 4-OH in the trans configuration points toward Glu674, whereas cis-4-OH points in the opposite direction toward catalytic Cys844. This difference results in different interactions with the enzyme. The trans-4-OH groups of THDP and THLP hydrogen bond with Glu674 (Figs. 4B and 4E), whereas the cis-4-OH of CHDP forms water-mediated hydrogen bonds with the backbone of the catalytic loop (Fig. 4C).

The hydrogen bonding of the trans-4-OH group seems to be more optimal in the D-isomer. In the THDP complex, Glu674 forms a bidentate hydrogen bond interaction with the 4-OH and the amino groups of the inhibitor (Fig. 4B). Note that the 4-OH and amino groups are perfectly spaced to engage the carboxylate of glutamate, and as result the donor-acceptor distances are fairly short: 2.5 Å and 2.8 Å. The interactions of THLP with Glu674 appear to be somewhat less favorable; although two hydrogen bonds are present, they involve a single O atom of Glu674, and the donor-acceptor distances are 0.3 Å longer than in THDP (Fig. 4E).

3.4. Remote binding site for THDP and D-Pro

The electron-density maps for the THDP and D-Pro structures revealed a possible secondary binding site. Strong electron density for THDP and D-Pro was observed in a location outside of the GSALDH active site, as shown for the former ligand in Fig. 5A. The remote site is located 24 Å from the center of the GSALDH active site (Fig. 1A) and consists of a shallow pocket on the surface of the NAD⁺-binding domain of the GSALDH module of SmPutA (Fig. 5B). The ligand in this location mainly interacts with the enzyme via its carboxylate group and an ordered water molecule (Fig. 5B). The remote site is available only in chain A of the dimer; the site in chain B is filled by side chains from a symmetry-related protein in the crystal lattice. Electron density for the other three ligands (L-Pro, THLP, and CHLP) in the remote site was not observed. Curiously, THDP and D-Pro were the only structures determined in the absence of NAD(H), raising the possibility of communication between the NAD⁺- and remote proline sites.

Fig. 5.

Remote binding site for THDP. (A) Polder omit electron density (4σ) and interactions for THDP bound to a remote site of the GSALDH domain. (B) Surface representation of the remote site.

3.5. In-solution analysis of hydroxyprolines binding to SmPutA

Thermal shift assays were used to confirm the binding of hydroxyprolines to SmPutA in solution (Fig. 6). The midpoint of the stability curve (T_1/2) of ligand-free SmPutA was 40.2 °C. All four hydroxyproline compounds (included at 62.5 mM) caused an increase in T_1/2, implying binding to the enzyme. THDP produced the largest increase of T_1/2 (5.6 °C), followed by the other D-isomer, CHDP (ΔT_1/2 = 4.0 °C). The shifts induced by THLP and CHLP were 2.9 °C and 2.1 °C, respectively. These data suggest that the D-hydroxyprolines are better at stabilizing SmPutA than L-hydroxyprolines, and the trans-isomer of each is more stabilizing than the cis-isomer.

Fig. 6.

Stability curves from fluorescence-based thermal shift assays with hydroxyproline compounds at 62.5 mM.

3.6. Inhibition of the GSALDH activity of SmPutA by prolines and hydroxyprolines

The GSALDH activity of SmPutA was measured with L-P5C as the variable substrate and NAD⁺ at fixed concentration. Since the proline compounds bind to the P5C/GSAL binding site, the data were fit initially to the competitive model (Fig. S1 of Supplementary data). This analysis suggested that CHLP does not inhibit the GSALDH activity of SmPutA, and the other compounds are weak inhibitors with inhibition constants in the range of 2 – 11 mM (Table 2). For reference, the K_m for L-P5C is 0.3 ± 0.1 mM. The quality of the fits to the competitive model was not optimal, so other inhibition models were tested. Improved fit quality was obtained when applying the uncompetitive inhibition model for D-proline and L-proline, and the mixed inhibition model for THDP, CHDP, and THLP (Fig. 7). The resulting inhibition parameters are in the millimolar range, consistent with these compounds being weak inhibitors (Table 2). We note that α is close to one for the mixed inhibitors, implying the uncompetitive component is significant for these compounds (Table 2).

Table 2.

Inhibition Constants from Various Models^a

Compound	Model	Ki (mM)	α	Adjusted R2
D-Pro	Competitive	1.5 ± 0.5	N/A	0.889
THDP	Competitive	4.5 ± 0.5	N/A	0.984
CHDP	Competitive	8. ± 1.	N/A	0.968
L-Pro	Competitive	11. ± 2.	N/A	0.941
THLP	Competitive	7. ± 1.	N/A	0.974
D-Pro	Uncompetitive	4.5 ± 0.2	N/A	0.992
THDP	Mixed	10. ± 1.	1.6 ± 0.4	0.996
CHDP	Mixed	25. ± 9.	0.9 ± 0.5	0.985
L-Pro	Uncompetitive	19. ± 2.	N/A	0.974
THLP	Mixed	27. ± 4.	0.6 ± 0.1	0.997

^aP5C was the variable substrate with NAD⁺ fixed at 0.2 mM.

Fig. 7.

Inhibition of the GSALDH activity of SmPutA by prolines and hydroxyprolines. The assays were performed at room temperature with NAD⁺ at 0.2 mM and SmPutA at 170 nM in a buffer containing 100 mM sodium phosphate pH 7 and 1 mM EDTA. The data for each inhibitor were analyzed by global fitting to the either the uncompetitive inhibition model (D-proline, L-proline) or the mixed inhibition model (THDP, CHDP, THDP) using Origin software. See Table 2 for inhibition constants.

4. Discussion

Although the inhibition of GSALDH by L-proline has been investigated previously, to our knowledge, the larger question of how D-proline and hydroxyprolines interact with the enzyme has not. L-proline was previously found to competitively inhibit (with P5C) mammalian GSALDH with K_i of 3 mM [13]. Similarly, the product L-glutamate, as well as L-glutamate analogs, inhibit mammalian GSALDH with millimolar competitive (with P5C) K_i [13, 27]. We showed here that the GSALDH active site also accommodates D-proline, THDP, CHDP, and THLP. The structures revealed a conserved mode of recognition of the carboxylate group, which results in the pyrrolidine rings of the D- and L-isomers having different orientations and different hydrogen bonding environments. We note that the interaction mode of the carboxylate group observed is similar to that of GSALDH with the product glutamate [28, 31].

We were unable to obtain a structure of the CHLP complex. The absence of electron density for CHLP when co-crystallized at 50 mM implies that this compound has very low affinity for the GSALDH site of SmPutA, consistent with the absence of measurable inhibition by CHLP in activity assays (Fig. 7). We rationalized this result by docking a model of CHLP into the active site based on the L-Pro complex structure (Fig. S2 of Supplementary data). The cis-4-hydroxyl of CHLP is predicted to clash with aromatic box residue Phe1010. Apparently, Phe1010 does not have sufficient conformational flexibility to relieve this steric clash. This idea is consistent with the observation that the conformation of Phe1010 is invariant in the five structures, compared to Phe708 (Fig. 3).

Kinetic data suggest that hydroxyprolines are weak inhibitors of the GSALDH activity of SmPutA. This result may reflect the high polarity of these compounds, which results in significant hydrogen bonding with water when free in solution. Binding to the enzyme likely requires a significant desolvation penalty, which may not be offset by the interactions observed in the crystal structures.

Although the hydroxyprolines are weak inhibitors, the kinetic data revealed a few trends. For example, the D-isomer of an enantiomeric pair binds more tightly than the L-isomer, based on activity and thermal shift measurements. The crystal structures are consistent with the solution data, showing that the D-isomers enjoy better stabilization of the carboxylate group due to a water molecule that is absent in the structures of the L-isomer complexes. A trend among the hydroxyprolines is that the trans configuration of the hydroxyl group results in better binding than the cis configuration, regardless of the stereochemistry around C1. The crystal structures also provide an explanation for this result: the hydroxyl group of THDP engages in geometrically optimal bidentate hydrogen bonding with Glu674 (Fig. 4B), whereas the cis-hydroxyl of CHDP hydrogen bonds with only a water molecule (Fig. 4C). Similarly, the hydroxyl of THLP engages Glu674 (Fig. 4E), whereas a predicted steric clash of the cis-hydroxyl of CHLP with Phe1010 strongly discourages binding (Fig. S2 of Supplementary data).

Curiously, the compounds did not exhibit classical competitive inhibition with P5C. This was unexpected, based on the observation that they occupy the GSAL binding site of SmPutA, as well as previous work showing that L-proline inhibits human GSALDH competitively with P5C [13]. The uncompetitive mode of inhibition observed here may be related to the fact that we used a bifunctional PRODH-GSALDH enzyme, whereas the human enzyme is a standalone, monofunctional GSALDH. A notable difference between these enzymes is that mammalian GSALDHs have a serine in the anchor loop in place of SmPutA Ala1003. Inhibition studies with other PutAs could help address this issue.

Uncompetitive inhibition is classically explained by a model in which the inhibitor binds to an enzyme-substrate complex. Examples of classical uncompetitive inhibition include caspase-6, low-molecular-weight protein tyrosine phosphatase, and alkaline phosphatase [32–34]. In these cases, crystal structures show the inhibitor bound in the active site alongside the substrate (or enzyme-substrate covalent intermediate). The structures show that the inhibitor forms noncovalent interactions with the substrate, providing clear explanations for the uncompetitive mode of inhibition.

The classic mechanism of uncompetitive inhibition is unlikely for the compounds studied here because the inhibitors and GSAL occupy the same site. We cannot exclude the possibility that the inhibitors occupy a different location when the substrate is bound. The remote site discovered in two of the structures is an intriguing candidate for such an alternative site (Fig. 5). We note the region harboring the remote site is structurally variable in PutAs and monofunctional GSALDHs. Therefore, if the remote site does play a role in inhibition, it may be limited to SmPutA.

Finally, Green et al. reported a similar conundrum for S-nitrosoglutathione reductase, where the crystal structure implies competitive inhibition, whereas the kinetic data suggest uncompetitive inhibition [35]. Like GSALDH, S-nitrosoglutathione reductase catalyzes an oxidoreductase reaction using NAD as the cofactor. The crystal structure revealed that inhibitor N6022 adopts the binding pose of the substrate S-nitrosoglutathione, yet the kinetic data clearly showed that inhibition by N6022 was uncompetitive with respect to this substrate. They postulated that the inhibition was due to N6022 binding to the E-NAD⁺ complex, a form of the enzyme to which the substrate S-nitrosoglutathione does not bind. GSALDH has a similar catalytic mechanism as S-nitrosoglutathione reductase, and the analogous uncompetitive inhibitory complex would be E-NADH-I (Fig. S3 of Supplementary data). Indeed, one of our crystal structures shows an inhibitor (L-proline) bound to the GSALDH-NADH complex. It is possible that inhibitor binding stabilizes the enzyme-NADH complex, slowing the release of the final product and regeneration of the free enzyme. Additional studies will be needed to fully understand the molecular basis of the inhibition of SmPutA by prolines. Our results provide another example of the pitfalls of inferring the mechanism of inhibition from crystal structures alone.

Highlights.

• Crystal structures of PutA complexed with proline stereoisomers and analogs

• First structure of GSALDH complexed with D-proline

• First structure of GSALDH complexed with hydroxyproline

• First structure of GSALDH complexed with NADH

• Inhibitors occupy the aldehyde binding site, but inhibition is not competitive

Abbreviations:

ALDH

aldehyde dehydrogenase

GSAL

L-glutamate-γ-semialdehyde

GSALDH

L-glutamate-γ-semialdehyde dehydrogenase

PRODH

proline dehydrogenase

P5C

Δ¹-pyrroline-5-carboxylate

PutA

proline utilization A

SmPutA

proline utilization A from Sinorhizobium meliloti

CHLP

cis-4-hydroxy-L-proline

CHDP

cis-4-hydroxy-D-proline

THLP

trans-4-hydroxy-L-proline

THDP

trans-4-hydroxy-D-proline