Crystal Structure of Binary and Ternary Complexes of Archaeal UDP-galactose 4-Epimerase-like l-Threonine Dehydrogenase from Thermoplasma volcanium

Background: The catalytic mechanism of UDP-galactose 4-epimerase-like l-threonine dehydrogenase (GalE-like l-ThrDH) is unknown.

Results: Four crystal structures of archaeal GalE-like l-ThrDH in the presence of NAD⁺, an inhibitor, and two substrates were determined.

Conclusion: Tyr¹³⁷ is essential for substrate binding and catalysis by the enzyme.

Significance: This study provides new insight into substrate recognition by GalE-like l-ThrDH.

Abstract

A gene from the thermophilic archaeon Thermoplasma volcanium encoding an l-threonine dehydrogenase (l-ThrDH) with a predicted amino acid sequence that was remarkably similar to the sequence of UDP-galactose 4-epimerase (GalE) was overexpressed in Escherichia coli, and its product was purified and characterized. The expressed enzyme was moderately thermostable, retaining more than 90% of its activity after incubation for 10 min at up to 70 °C. The catalytic residue was assessed using site-directed mutagenesis, and Tyr¹³⁷ was found to be essential for catalysis. To clarify the structural basis of the catalytic mechanism, four different crystal structures were determined using the molecular replacement method: l-ThrDH-NAD⁺, l-ThrDH in complex with NAD⁺ and pyruvate, Y137F mutant in complex with NAD⁺ and l-threonine, and Y137F in complex with NAD⁺ and l-3-hydroxynorvaline. Each monomer consisted of a Rossmann-fold domain and a C-terminal catalytic domain, and the fold of the catalytic domain showed notable similarity to that of the GalE-like l-ThrDH from the psychrophilic bacterium Flavobacterium frigidimaris KUC-1. The substrate binding model suggests that the reaction proceeds through abstraction of the β-hydroxyl hydrogen of l-threonine via direct proton transfer driven by Tyr¹³⁷. The factors contributing to the thermostability of T. volcanium l-ThrDH were analyzed by comparing its structure to that of F. frigidimaris l-ThrDH. This comparison showed that the presence of extensive inter- and intrasubunit ion pair networks are likely responsible for the thermostability of T. volcanium l-ThrDH. This is the first description of the molecular basis for the substrate recognition and thermostability of a GalE-like l-ThrDH.

Introduction

l-Threonine dehydrogenase (l-ThrDH,² EC 1.1.1.103) catalyzes NAD⁺-dependent dehydrogenation at the β-carbon (C3) position of l-threonine. The suspected product of the reaction is l-2-amino-3-oxobutyrate, which nonenzymatically decomposes into aminoacetone and CO₂ (1) or is further cleaved in a CoA-dependent reaction to produce glycine and acetyl-CoA (2, 3). This is in contrast to other amino acid dehydrogenases such as glutamate or alanine dehydrogenase (4), which catalyze dehydrogenation at the α-carbon (C2) position of the amino acid, accompanied by deamination. In this respect, l-ThrDH can be regarded as a type of alcohol dehydrogenase. The reaction catalyzed by l-ThrDH is the first in the l-threonine degradation pathway and is widely distributed among eukaryotes, bacteria, and archaea (5–8). The primary structure of l-ThrDH was initially determined using the enzyme from Escherichia coli (9). The structure revealed the enzyme to be a member of the medium chain dehydrogenase reductase family. Subsequent analysis of the crystal structures of two other l-ThrDHs from the hyperthermophilic archaea Pyrococcus horikoshii and Thermococcus kodakaraensis showed that the overall fold of the monomer and the assembly of the tetramer were similar to those of alcohol dehydrogenases (10, 11).

We recently determined the crystal structure of an l-ThrDH from the psychrophilic bacterium Flavobacterium frigidimaris KUC-1 in complex with NAD⁺ and a substrate analog, glycerol (PDB code 2YY7) (12). This enzyme exhibited notable sequence identity with UDP-galactose 4-epimerase (GalE) homologues, and its overall fold showed significant similarity to those of GalEs, which belong to the short chain dehydrogenase reductase family. Thus F. frigidimaris l-ThrDH is markedly different from conventional l-ThrDHs, which belong to the medium chain dehydrogenase reductase family (12, 13). Structural comparison of F. frigidimaris l-ThrDH with E. coli and human GalEs showed a topological difference in three loops around the substrate and NAD⁺ binding site; in F. frigidimaris l-ThrDH, loops 1 and 2 insert toward the active site cavity, creating a barrier that prevents the binding of UDP-glucose, and the NAD⁺ binding loop, which tightly holds the adenine ribose moiety of NAD⁺ in the E. coli and human GalEs, is absent in the F. frigidimaris l-ThrDH (12, 14–16). By contrast, we found that the two signature sequences in the GalE, a YXXXK motif in which the conserved tyrosine plays a key role in catalysis, and a GXXGXXG motif, which is located near the cofactor binding pocket, are strictly conserved in the F. frigidimaris enzyme. This suggests the reaction catalyzed by F. frigidimaris l-ThrDH is probably similar to that catalyzed by GalE. Notably, however, the catalytic residue and the mechanism underlying the catalytic reaction of F. frigidimaris l-ThrDH remain uncertain due to the enzyme instability.

Within the genomic sequence of the thermophilic archaeon Thermoplasma volcanium we found a gene (open reading frame identification number TVN0454) whose predicted amino acid sequence exhibits 44% identity with that of F. frigidimaris l-ThrDH. In this study we expressed the gene, characterized the enzyme produced, and show that the enzyme is a thermostable GalE-like l-ThrDH. We also determined four crystal structures for this enzyme at 1.77–2.07 Å resolution in the presence of NAD⁺, an inhibitor, and two substrates. This is the first description of the molecular basis of the substrate recognition and thermostability of an archaeal GalE-like l-ThrDH. Our findings provide new insight into the catalytic mechanism of GalE-like l-ThrDH.

EXPERIMENTAL PROCEDURES

Cloning and Protein Expression

The gene encoding l-ThrDH (ORF ID: TVN0454, the gene information is available in the Kyoto Encyclopedia of Genes and Genomes (KEGG) database) was amplified by PCR. The oligonucleotide primers used to amplify the l-ThrDH gene fragment were 5′-TGATAACATATGATCCTAGTTACCGGCTCA-3′, which contains a unique NdeI restriction site overlapping the 5′ initiation codon, and 5′-AAGGAAGGATCCTCAGAGGGCATGTTTACC-3′, which contains a unique BamHI restriction site proximal to the 3′ end of the termination codon. Chromosomal T. volcanium DNA was isolated as described previously (17) and used as the template. The amplified 1.0-kb fragment was digested with NdeI and BamHI and ligated with the expression vector pET11a (Novagen) linearized with NdeI and BamHI to generate pETVN0454, which was then used to transform E. coli strain BL21 (DE3) codon plus RIL (Stratagene). The transformants were cultivated at 37 °C in 100 ml of Luria-Bertani medium until the optical density at 600 nm reached 0.6. Expression was then induced by adding 1.0 mm isopropyl-β-d-thiogalactopyranoside to the medium, and cultivation was continued for an additional 3 h at 37 °C.

Site-directed Mutagenesis

Site-directed mutagenesis was accomplished using a QuikChange XL site-directed mutagenesis kit (Stratagene) according to the manufacturer's instructions. pETVN0454 served as the template, and the following set of oligonucleotide primers was used as the mutagenic primers (the mutations are underlined): 5′-CCAAGGACAATGTTTGGTGTAACAAAA-3′ and 5′-TTTTGTTACACCAAACATTGTCCTTGG-3′ for Y137F, 5′-AACCCGGCCAAGCACAATGTATGGTGTAAC-3′ and 5′-GTTACACCATACATTGTGCTTGGCCGGGTT-3′ for R134S, and 5′-CTCGGGCAGTACTATTATAATAAATTTGGC-3′ and 5′-GCCAAATTTATTATAATAGTACTGCCCGAG-3′ for E153N.

Purification of Recombinant l-ThrDH

All of the purification steps were carried out at room temperature. E. coli cells harvested from the 100 ml of culture (about 1.0 g (wet weight)) were used as the starting material for purification of l-ThrDH. To prepare the crude extract, the cells were washed twice and suspended in 10 mm potassium phosphate buffer (pH 7.0). The cells were then disrupted by sonication and centrifuged at 20,000 × g for 10 min. The resultant supernatant, which served as the crude extract, was heated at 70 °C for 10 min in the presence of 0.2 m Na₂SO₄ (except for the R134S and E153N mutant enzymes), and the denatured proteins were removed by centrifugation (20,000 × g for 10 min). The supernatant from that step was dialyzed against 10 mm potassium phosphate buffer (pH 7.0), and the remaining enzyme solution was applied to a Red-Sepharose CL-4B column (10 × 110 mm) equilibrated with 10 mm potassium phosphate buffer (pH 7.0). After washing the column with the same buffer, the enzyme was eluted with a 100-ml linear gradient of 0–0.5 m NaCl in the same buffer. The active fractions were pooled, and the enzyme solution was dialyzed against 10 mm potassium phosphate buffer (pH 7.0) containing 50 mm NaCl.

Determination of Enzyme Activity

Enzyme activity was assayed spectrophotometrically using a Hitachi U-2910 spectrophotometer equipped with a thermostat. The standard reaction mixture for oxidation consisted of 100 mm glycine-NaOH buffer (pH 10.0) containing 25 mm l-threonine, 1.25 mm NAD⁺, and the enzyme in a final volume of 1.0 ml. The pH values of the buffer and threonine solutions were adjusted at room temperature and used for the assay without temperature compensation. After warming the reaction mixture by incubation for 3 min at 50 °C without the coenzyme, the reaction was started by adding the coenzyme. The appearance of NADH was monitored from the absorbance at 340 nm (extinction coefficient ϵ = 6.22 mm⁻¹·cm⁻¹). One unit (U) of activity was defined as the amount of enzyme producing 1.0 μmol of NADH per min at 50 °C under the standard assay conditions, and the specific activity was expressed in U·(mg of protein)⁻¹. The protein concentration was determined using the Bradford method; bovine serum albumin served as the standard (18). The enzyme activity of GalE was assayed as previously described (13).

Polyacrylamide Gel Electrophoresis and Molecular Mass Determination

SDS-PAGE (12% acrylamide slab gel, 1-mm thick) was carried out using the procedure of Laemmli (19), after which the protein band was stained with Coomassie Brilliant Blue R-250.

The molecular mass of the purified enzyme was determined using an analytical Superose 6 (GE Healthcare) gel filtration column previously equilibrated with 10 mm potassium phosphate buffer (pH 7.0) containing 0.2 m NaCl. Tyroglobulin (molecular mass, 669 kDa), ferritin (440 kDa), aldolase (158 kDa), albumin (67 kDa), ovalbumin (43 kDa), chymotrypsinogen A (25 kDa), and ribonuclease A (13.7 kDa) supplied in gel filtration calibration kits (GE Healthcare) were used as molecular mass standards. The subunit molecular mass of the enzyme was determined by SDS-PAGE.

Analysis of N-terminal Amino Acid Sequences

Approximately 3 μg of the purified enzyme were subjected to SDS-PAGE as described above and then electroblotted onto a polyvinylidene difluoride membrane, after which the membrane was stained with Coomassie Brilliant Blue R-250 and destained. To determine the N-terminal amino acid sequences, the protein band was then subjected to automated Edman degradation using a PPSQ-21A Protein Sequencer (Shimadzu).

Stability, pH Optimum, and Kinetic Parameters

To determine the effect of temperature on its stability, the enzyme was incubated for 10 min at different temperatures in 10 mm potassium phosphate buffer (pH 7.0) containing 50 mm NaCl. The residual activity was determined using the standard assay method. To determine the effect of pH on its stability, the enzyme was incubated for 20 min in buffer at different pH values (50 °C), and the remaining activity was again determined using the standard assay method. The buffers (100 mm) used for these assays were citrate (pH 3.0–6.0), potassium phosphate (pH 6.0–7.5), Tris-HCl (pH 7.5–9.0), and glycine-NaOH (pH 9.0–11.5). The same buffers were also used to determine the optimal pH for enzyme activity at 50 °C. The initial velocity was determined by varying the concentration of one substrate while keeping the concentrations of the other substrates constant, as previously described (20).

Crystallization and Data Collection

Initial screening for crystallization was carried out using Cryo 1 and 2 (Emerald Biostructures) at 20 °C using the sitting-drop vapor diffusion method. Crystals of the wild-type and Y137F mutant enzymes were grown in sitting drops composed of 1 μl of enzyme solution (wild type, 40 mg/ml; Y137F, 25 mg/ml) containing 1 mm NAD⁺ mixed with 1 μl of mother liquor containing 100 mm cacodylate buffer (pH 6.4), 50% (v/v) 2-methyl-2,4-pentandiol (MPD), and 5% (w/v) poly(ethylene glycol) 8000. Crystals of NAD⁺/pyruvate-bound wild-type enzyme were prepared by soaking the crystals in reservoir solution containing 0.3 m pyruvate for 2 h. Crystals of NAD⁺/l-threonine-bound and NAD⁺/l-3-hydroxynorvaline-bound Y137F mutant enzyme were prepared by soaking the crystals in reservoir solution containing 0.1 m l-threonine for 8 h or 0.1 m dl-3-hydroxynorvaline for 7 h. Data were collected using an ADSC CCD detector system on the BL-5A, BL-17A, AR-NW12A, and AR-NE3A beamlines at the Photon Factory in Tsukuba, Japan. All diffraction measurements were carried out on crystals cryoprotected with 50% (v/v) MPD and cooled to 100 K in a stream of nitrogen gas. The data were processed using HKL2000 (21).

Phasing and Refinement

The initial phases of the wild-type enzyme structure were determined by molecular replacement using the program MOLREP in the CCP4 program suite (22); the structure of chain A of l-ThrDH from F. frigidimaris (Protein Data Bank entry 2YY7) served as the search model. Further model building was performed with the program COOT (23), and refinement to a resolution of 2.07 Å was carried out using Refmac5 (24) and CNS (25). The NAD⁺ molecule was clearly visible in both the σ_A-weighted 2 F_o − F_c and F_o − F_c density maps and was included in the latter part of the refinement. Model geometry was analyzed using MOLPROBITY (26). Based on the model of the wild-type enzyme, the structures of the NAD⁺/pyruvate-bound wild-type (1.78 Å), NAD⁺/l-threonine-bound Y137F (1.85 Å), and NAD⁺/l-3-hydroxynorvaline-bound Y137F (1.77 Å) enzymes were refined using difference Fourier maps. Two NAD⁺ molecules and two MPD molecules were included in the refinement. The data collection and refinement statistics are listed in Table 1.

TABLE 1.

Statistics for data collection, phase determination, and refinement

	Wild-type/NAD+	Wild-type/NAD+/pyruvate	Y137F/NAD+/l-threonine	Y137F/NAD+/l-3-hydroxynorvaline
Data collection
Space group	C2	C2	C2	C2
Unit cell parameters	a = 149.7 Å	a = 149.8 Å	a = 150.2 Å	a = 150.5 Å
	b = 45.6 Å	b = 45.9 Å	b = 46.3 Å	b = 46.5 Å
	c = 89.0 Å	c = 89.7 Å	c = 89.7 Å	c = 89.9 Å
	β = 113.1	β = 113.3	β = 113.2	β = 113.2
Wavelength (Å)	1.0	1.0	1.0	1.0
X-ray source	BL-5A	BL-17A	AR-NW12A	AR-NE3A
Maximum resolution (Å)	2.07	1.78	1.85	1.77
No. of unique reflections	33,910	53,908	48,200	54,564
Redundancy	5.1	5.5	7.5	5.6
Completeness (%)a	99.8 (99.6)	99.3 (90.0)	99.7 (99.3)	97.5 (95.0)
Rsyma,b (%)	6.7 (28.3)	5.1 (12.4)	6.0 (26.9)	4.8 (14.6)
〈I/σ (I)〉c	15.2	23.8	15.4	25.2
Refinement
Resolution range (Å)	32.3–2.07	43.8–1.78	32.1–1.85	33.9–1.77
Rcrystd (%)	21.5	20.2	19.1	19.9
Rfreee (%)	25.7	21.8	20.9	22.1
No. of protein atoms	4984	4944	4982	4946
No. of water molecules	258	296	247	243
No. of ligands	NAD+, 2	NAD+, 2	NAD+, 2	NAD+, 2
		MPD, 2	MPD, 2	MPD, 2
		Pyruvate, 2	l-Threonine, 2	l-3-Hydroxynorvaline, 2
r.m.s.d. bond lengths (Å)	0.006	0.006	0.006	0.006
r.m.s.d. bond angles (°)	1.2	1.3	1.2	1.2
Average B-factors (Å2)
Protein atoms	32.5	20.5	21.0	23.6
Water molecules	39.5	27.4	27.2	30.4
NAD+ molecules	31.7	18.5	38.1	38.4
MPD molecules		33.0	36.9	37.4
Pyruvate molecules		21.2
l-Threonine molecules			25.7
l-3-Hydroxynorvaline molecules				20.3
Ramachandran quality f
Favored regions (%)	96.3	96.3	97.1	96.4
Outliers (%)	0.5	0.3	0	0.3
PDB accession codes	3A1N	3A4V	3A9W	3AJR

^a Values in parentheses are for the last resolution shell.

^b R_sym = Σ_hΣ_i|I_i(h) − 〈I(h)〉|/Σ_hΣ_i| I_i(h)|, where I_i (h) is the intensity measurement for a reflection h, and 〈I(h)〉 is the mean intensity for this reflection.

^c For the highest resolution shell, the number of the reflections with I/σ(I) less than 1 was 293 (18.0%), 158 (5.8%), 329 (13.8%), and 351 (12.6%) for wild-type/NAD⁺, wild-type/NAD⁺/pyruvate, Y137F/NAD⁺/l-threonine, and Y137F/NAD⁺/l-3-hydroxynorvaline data, respectively.

^d R_cryst = Σ_h‖F_obs| −|F_calc‖/Σ_h|F_obs|.

^e R_free was calculated using randomly selected reflections (10%).

^f MOLPROBITY (26) was used to monitor and validate the structural model.

Structure Analysis

Hydrogen bonds were identified using the program CCP4 mg (27). Ion pairs (the cut-off distance was 4.0 Å (28)) were identified using the WHAT IF web server (29). Molecular graphics were created using PyMOL.

RESULTS AND DISCUSSION

Gene Expression and Enzyme Purification

Within the sequence of the T. volcanium genome, we identified a gene, TVN0454 (954 bp at position 440460–441413 of the entire genome) whose predicted amino acid sequence showed 44% identity with that of F. frigidimaris l-ThrDH. After transforming E. coli with pETVN0454, an expression vector harboring the gene, the transformant cells exhibited a high level of l-ThrDH activity, and the enzyme was readily purified from the cell crude extract in two simple steps: heat treatment and affinity chromatography of NAD(P)⁺-binding proteins. About 15 mg of the purified enzyme was obtained from 1 g (wet weight) of cells. The purified enzyme showed a single protein band on SDS-PAGE, and its N-terminal sequence was determined to be MILVTGSSGQ, which corresponds to that predicted from the TVN0454 gene sequence and confirms that TVN0454 encodes the T. volcanium l-ThrDH. The specific activity of the purified wild-type and mutant enzymes was estimated to be about 40.1 U·mg⁻¹ (wild-type), 0.2 U·mg⁻¹ (Y137F), 37.1 U·mg⁻¹ (R134S), and 36.6 U·mg ⁻¹ (E153N) at 50 °C.

Molecular and Catalytic Properties of T. volcanium l-ThrDH

SDS-PAGE showed the subunit molecular mass of T. volcanium l-ThrDH to be about 35 kDa, which is consistent with the molecular weight (35,843) calculated from the amino acid sequence. That the native molecular mass determined by gel filtration was about 77 kDa suggests the native enzyme is a homodimer. Evaluation of the catalytic activity (oxidation of l-threonine) at temperatures ranging from 20 to 100 °C revealed the enzyme to be maximally active at around 70 °C. The enzyme was also somewhat thermostable, retaining more than 90% of its activity after incubation for 10 min at temperatures up to 70 °C (supplemental Fig. 1). Evaluation of the catalytic activity at different pH values revealed the enzyme to be maximally active at around pH 10.0. The enzyme was stable over a wide range of pH values, losing no activity when incubated at pH values between 4.5 and 9.5 for 20 min at 50 °C.

We next assessed the electron donor specificity for oxidation catalyzed by T. volcanium l-ThrDH. With NAD⁺ (1.25 mm) as the electron acceptor, the enzyme acted on l-threonine and dl-3-hydroxynorvaline (25 mm substrate), and the activity with dl-3-hydroxynorvaline was 48% of that seen with l-threonine. Inert were d-threonine, dl-allo-threonine, dl-threo-3-phenylserine, l-serine, l-homoserine, l-aspartate, l-glutamate, l-methionine, l-alanine, l-leucine, l-phenylalanine, l-proline, glycine, l-lysine, l-asparagine, l-arginine, l-isoleucine, l-histidine, l-valine, l-tryptophan, 2-propanol, 1,4-butanediol, 2,3-butanediol, 1,2-propanediol, ethanol, 1-butanol, pyruvate, and d-glucose. T. volcanium l-ThrDH was highly specific for NAD⁺ as the electron acceptor and was not active with NADP⁺. In addition, we found that T. volcanium l-ThrDH did not show GalE activity. The enzyme's high specificity for l-threonine as well as its relatively high stability would be advantageous for enzymatic detection of l-threonine (30) or for use as an l-threonine biosensor.

We also examined the effect of various chemicals on the enzyme activity. T. volcanium l-ThrDH was unaffected by EDTA, LiSO₄, MgCl₂, MnCl₂, CaCl₂, NiCl_2, CoCl_2, BaCl₂, HgCl₂, CdSO₄, CuSO₄, ZnCl₂, or iodoacetic acid (each at 1 mm). On the other hand, the enzyme was partially inhibited by pyruvate, retaining 80% of its activity in the presence of 10 mm pyruvic acid sodium salt. This is consistent with the earlier observation that pyruvate acts as a competitive inhibitor of l-threonine (K_i = 23.2 mm) with the F. frigidimaris l-ThrDH (13). T. volcanium l-ThrDH showed typical Michaelis-Menten kinetics; for oxidation, the K_m values for NAD⁺ and l-threonine were 0.185 and 17.5 mm, respectively.

Overall Structures

The structure of the NAD⁺-bound wild-type T. volcanium l-ThrDH was determined using molecular replacement and was refined at a resolution of 2.07 Å to a crystallographic R-factor of 21.5% and a free R-factor of 25.7% (Table 1). The asymmetric unit consisted of one homodimer with a solvent content of 37%, which corresponds to a Matthew's coefficient (31) of 2.0 ųDa⁻¹. The model contained 315 ordered amino acid residues in each subunit and 258 water molecules. The two nearly identical (r.m.s.d. = 0.12 Å) subunits had approximate dimensions of 52 × 32 × 36 Å, were related by a 2-fold noncrystallographic rotation axis, and were closely associated through four helices (α5 and α7 in each subunit) and two loops (Val¹¹⁵–Met¹³⁶ in each subunit) (Fig. 1A). Each monomer consisted of two domains, an N-terminal coenzyme-binding domain (α1-α7, α9-α10, α13, β1-β6, and β10), which formed a classical Rossmann-fold motif, including two dimerization helices, and a C-terminal domain (α8, α11, α12, α14, β7-β9, and β11-β12) in which the catalytic activity was mediated. The model of the NAD⁺/pyruvate-bound wild-type enzyme was refined at a resolution of 1.78 Å to a crystallographic R-factor of 20.2% and a free R-factor of 21.8% (Table 1). The final model was composed of amino acid residues 1–311, 2 NAD⁺ coenzymes, 2 MPDs, 2 pyruvates, and 296 waters. The model of the NAD⁺/l-threonine-bound Y137F mutant enzyme was refined at a resolution of 1.85 Å to a crystallographic R-factor of 19.1% and a free R-factor of 20.9% (Table 1). The final model was composed of amino acid residues 1–315, 2 NAD⁺ coenzymes, 2 MPDs, 2 l-threonines, and 247 waters. The model of the NAD⁺/l-3-hydroxynorvaline-bound Y137F mutant enzyme was refined at a resolution of 1.77 Å to a crystallographic R-factor of 19.9% and a free R-factor of 22.1% (Table 1). The final model was composed of amino acid residues 1–314, 2 NAD⁺ coenzymes, 2 MPDs, 2 l-3-hydroxynorvalines, and 243 waters. In all cases the C-terminal residues (Aal³¹⁶–Leu³¹⁷) were disordered and not visible in the electron-density map.

FIGURE 1.

Overall structure and structural comparison of T. volcaniuml-ThrDH. A, shown is a ribbon representation of the T. volcanium l-ThrDH dimer. The NAD⁺ binding and catalytic domains are shown in cyan and purple, respectively. The adjacent subunit is shown in gray. The region involved in dimer formation is indicated. B, superimposed Cα traces of the NAD⁺ binding domains are shown. T. volcanium l-ThrDH/NAD⁺ is in green, T. volcanium l-ThrDH/NAD⁺/pyruvate is in magenta, and F. frigidimaris l-ThrDH is in blue. NAD⁺ (yellow) and pyruvate (white) molecules are shown as stick models. Oxygen and nitrogen atoms are shown in red and blue, respectively. C, shown are superimposed Cα traces of the catalytic domains. l-ThrDHs are colored as in B.

The main chain coordinates of the T. volcanium l-ThrDH/NAD⁺ and l-ThrDH/NAD⁺/pyruvate monomers were basically the same as the F. frigidimaris l-ThrDH monomer (r.m.s.d. = 1.1–1.2 Å for the Cα atoms of 306 residues; PDB code 2YY7) (Fig. 1, B and C), as was assembly of the dimer. In addition, there was no apparent conformational difference among the four T. volcanium l-ThrDH structures studied (r.m.s.d. = 0.38–0.41 Å for the Cα atoms of 311–315 residues), which suggests substrate binding does not affect the mutual orientation of the two domains of the T. volcanium enzyme. The gross movements might be precluded by crystal packing.

Structural Features Underlying Thermostability

F. frigidimaris l-ThrDH rapidly inactivates at 50 °C (13) and is thus much less stable than the T. volcanium enzyme. Therefore, the factors contributing to the relative thermostability of T. volcanium l-ThrDH were analyzed by comparing its structure to that of the F. frigidimaris enzyme. Structural studies of hyperthermophilic proteins have shown that the number of ion pairs and the formation of ion pair networks contribute significantly to the thermostability of these enzymes (32–34). Using a cutoff distance of 4.0 Å between oppositely charged residues, we calculated that T. volcanium l-ThrDH contains 26 intrasubunit ion pairs, whereas F. frigidimaris l-ThrDH contains only 13 intrasubunit ion pairs. In addition, two major intersubunit ion pair networks between the A and B subunits were observed in T. volcanium l-ThrDH: Arg¹³²–Glu¹¹⁹–Arg¹³⁴–Glu¹⁵³′ and Arg¹³²′–Glu¹¹⁹′–Arg¹³⁴′–Glu¹⁵³ (the primes indicate the neighboring subunit in the dimer) (Fig. 2A). In F. frigidimaris l-ThrDH, however, the charged Glu¹¹⁹, Arg¹³², Arg¹³⁴, and Glu¹⁵³ are replaced by Thr¹²⁵, Glu¹³⁸, Ser¹⁴⁰, and Asn¹⁵⁹, respectively (Fig. 2B). Because Arg¹³⁴ is involved in both inter- and intrasubunit ion pairs and Glu¹⁵³ is involved with intersubunit ion pairs (Fig. 2A), we constructed two T. volcanium l-ThrDH single point mutants (R134S and E153N) and compared their thermostability with that of the wild-type enzyme. We found that the mutant enzymes were significantly less thermostable, with both mutants retaining full activity only at 50 °C or less (supplemental Fig. 1). This suggests the presence of the extensive inter- and intrasubunit ion pair networks is likely responsible for the thermostability of T. volcanium l-ThrDH.

FIGURE 2.

The largest intersubunit ion pair network, formed by four residues in T. volcaniuml-ThrDH (A) and the equivalent positions in F. frigidimarisl-ThrDH (B). l-ThrDHs are colored as in Fig. 1B. The adjacent subunit is shown in gray.

Active Site and l-Threonine Binding Model

Within all four T. volcanium l-ThrDH structures studied, the electron density corresponding to the NAD⁺ coenzyme bound within the active site was very clear, which enabled us to place the ligand with reasonable accuracy (supplemental Fig. 2). Moreover, in the initial electron density map of the NAD⁺/pyruvate-bound wild-type enzyme, we observed an extra density within the active site cavity, and after construction and refinement of the peptide chain, a pyruvate molecule could be modeled into that density. However, we could not determine precise orientation of the pyruvate molecule, as pyruvate is an approximately symmetrical molecule. In our predictive model (Fig. 3A), two oxygen atoms of the C1 carboxylate are situated within hydrogen bonding distance of the side chains of Ser⁷⁴, Thr¹¹², and Tyr¹³⁷ and the main chain amide proton of Ser⁷⁴. The oxygen atoms of the C2 carbonyl group are within hydrogen bonding distance of the side chain of Ser⁷⁴, a proton in the main chain amide of Thr¹⁷⁸, and a water molecule (WAT 427). Among these residues, Tyr¹³⁷ is strictly conserved in GalE-like l-ThrDHs and GalEs. As mentioned above, Y137F mutation nearly abolished the activity of T. volcanium l-ThrDH, which suggests Tyr¹³⁷ plays a critical role in the catalytic reaction of GalE-like l-ThrDHs (see below).

FIGURE 3.

Stereo view of the active site of T. volcaniuml-ThrDH. A, the NAD⁺/pyruvate-bound wild-type enzyme (magenta) is shown. B, the NAD⁺/l-threonine-bound Y137F mutant enzyme (white) is shown. C, the NAD⁺/l-3-hydroxynorvaline-bound Y137F mutant enzyme (gold) is shown. D, superposition of the active site pocket of the NAD⁺/l-threonine-bound Y137F mutant with that of the NAD⁺/l-3-hydroxynorvaline-bound Y137F mutant is shown. For clarity, the Tyr¹³⁷ residue was positioned based on the orientation of Phe¹³⁷ in Y137F. Atoms are colored as in Fig. 1. The C4 atom of the pyridine ring (a hydride acceptor site) and the si- and re-faces are labeled. The l-threonine and l-3-hydroxynorvaline molecules are shown as stick models in white. WAT, water. In panels A–C, the final σ_A-weighted F_o − F_c omit electron density maps for the ligands are shown at the 1σ level.

Given the structure of pyruvate, we sought to predict the structure of the l-threonine molecule within the active site of T. volcanium l-ThrDH. Based on the structure of pyruvate, we tried the modeling of l-threonine binding into the active site of T. volcanium l-ThrDH. However, we could not postulate a single binding mode of l-threonine molecule. On the other hand, using the inactive T. volcanium l-ThrDH Y137F mutant, which trapped the substrate within the active site, we were able to determine the substrate-bound structures of the enzyme in complex with l-threonine or l-3-hydroxynorvaline. In both cases, the map clearly defined the precise orientation of the l-threonine and l-3-hydroxynorvaline (Fig. 3, B and C); two oxygen atoms of the Cα carboxylate are situated within hydrogen bonding distance of the side chains of Ser⁷⁴ and Thr¹⁷⁹ and the main chain amide proton of Ser⁷⁴, Thr¹⁷⁸, and Thr¹⁷⁹. The oxygen atoms of the Cβ hydroxyl group are within hydrogen bonding distance of the side chain of Thr¹¹². In addition, the Cα amino group is within hydrogen bonding distance of the side chain of Thr¹⁷⁹, the carbonyl group of the nicotinamide ring, and a water molecule. All of the residues are conserved in F. frigidimaris l-ThrDH, except Thr¹¹² is replaced by a homologous serine residue (Fig. 4).

FIGURE 4.

Structure-based amino acid sequence alignment of l-ThrDHs and GalEs (Tvo LTDH, T. volcaniuml-ThrDH (PDB code 3A4V); Ffr LTDH, F. frigidimarisl-ThrDH (PDB code 2YY7); Eco GalE, E. coli GalE (PDB code 1XEL); Hum GalE, human GalE (PDB code 1EK6)). Sequences were aligned using DALI (39). The GXXGXXG and YXXXK motifs are underlined. Black boxes represent loops responsible for substrate recognition (Loop 1 and Loop 2) and NAD⁺ binding (NAD⁺ binding loop). The residues involved in l-threonine binding are shown in blue boxes. The conserved Thr/Ser-Tyr-Lys catalytic triad was shown in red boxes. The GTTDY motif is shown in cyan. Asterisks indicate conserved residues. The α-helices (α1-α14; yellow) and β-strands (β1-β12; purple) in T. volcanium l-ThrDH are shown above the alignment.

Insight into ThrDH Reaction

With short chain dehydrogenase reductase family enzymes, the Thr/Ser-Tyr-Lys catalytic triad is thought to be important for catalysis. The side chain oxygen of the tyrosine residue functions as an acid-base catalyst for proton transfer, and the serine residue plays a subsidiary role for stabilization of substrate binding (35). In addition, the lysine residue has two critical functions; it interacts with the hydroxyl groups of the nicotinamide ribose (cofactor binding), and it lowers the pK_a value of the hydroxyl group in the side chain of the tyrosine residue (35). In the E. coli GalE, it has been reported that the mutation of Lys¹⁵³ to Met or Ala abolishes the UMP dependence of reductive inactivation by sodium cyanoborohydride. This suggests that the positive electrostatic field would stabilize the phenolate form of Tyr¹⁴⁹ and thereby account for a low value of pK_a (14). This Thr/Ser-Tyr-Lys catalytic triad is strictly conserved in E. coli and human GalEs and F. frigidimaris and T. volcanium l-ThrDHs (Fig. 4). Thus, a common catalytic mechanism should be predictable among short chain dehydrogenase reductase family enzymes.

We recently proposed two possible catalytic mechanisms for F. frigidimaris l-ThrDH based on the l-threonine binding models deduced from the structures of bound glycerol and the glucose moiety of the UDP-glucose in the E. coli and human GalEs (12). Based on the model deduced from the structure of the glycerol-bound enzyme, we proposed that the reaction proceeds through a proton shuttle mechanism in which Tyr¹⁴³ provides the driving force for general acid-base catalysis and Ser¹¹⁸ mediates the proton relay as in the case of E. coli GalE (36, 37). With that mechanism, the conserved Tyr¹⁴³ cannot function as the active-site base because the observed distance (4.9 Å) between the Oη of Tyr¹⁴³ and the β-hydroxyl group of the l-threonine is too large for direct interaction. By contrast, based on the l-threonine binding model deduced from the structure of UDP-glucose, Tyr¹⁴³ lies within hydrogen-bonding distance (2.7 Å) of the β-hydroxyl group of the l-threonine, so that this tyrosine residue could serve directly as the active-site base as in the case of human GalE (16). At that time, we could not determine which was the correct catalytic mechanism of F. frigidimaris l-ThrDH.

Within the structures of the T. volcanium l-ThrDH Y137F/NAD⁺/l-threonine and l-3-hydroxynorvaline, we observed that the Oη of Tyr¹³⁷, which corresponds to Tyr¹⁴³ in F. frigidimaris l-ThrDH, lies within hydrogen-bonding distance (2.4–2.6 Å) of the β-hydroxyl group of the l-threonine or l-3-hydroxynorvaline (Fig. 3D). This means that Tyr¹³⁷ could serve directly as the active-site base. In addition, the distance between the Oγ of Thr¹¹² and the β-hydroxyl of the l-threonine (2.8 Å) or l-3-hydroxynorvaline (2.8 Å) as well as the distance between the side chain hydroxyls of Thr¹¹² and Tyr¹³⁷ (3.0–3.2 Å) are in good agreement with those observed in the UDP-glucose based model of F. frigidimaris l-ThrDH. Taken together, these findings suggest the catalytic mechanism underlying the T. volcanium l-ThrDH reaction likely proceed through the following steps: 1) abstraction of the β-hydroxyl hydrogen of l-threonine via direct proton transfer driven by Tyr¹³⁷ and 2) transfer of a hydrogen from the β-carbon (C3) of the l-threonine to C4 of the NAD⁺ (si-face), forming l-2-amino-3-oxobutyrate and NADH (Fig. 5).

FIGURE 5.

Proposed catalytic mechanism of T. volcaniuml-ThrDH.

Distribution of GalE-like l-ThrDH Homologues

When we searched the genome data base using PSI-BLAST (38), we found that the amino acid sequence of the T. volcanium l-ThrDH exhibited relatively high identity with GalE-related proteins from archaea and bacteria: Thermoplasma acidophilum (Ta1111, 76%), Aciduliprofundum boonei (Aboo_0732, 53%), Desulfurococcus kamchatkensis (DKAM_0817, 53%), Candidatus Korarchaeum (Kcr_0631, 50%), and Thermosphaera aggregans (Tagg_1094, 50%) in archaea as well as Bacillus anthracis (BA_0621, 50%), Flavobacterium johnsoniae (Fjoh_2087, 46%), Staphylococcus aureus (SA0511, 45%), and Bacteroides thetaiotaomicron (BT_1370, 44%) in bacteria. Moreover, we also found GalE-like l-ThrDH homologues in mosquito (AgaP_AGAP011948, 45%), fruit fly (Dmel_CG5955, 44%), chimpanzee (463989, 43%), rat (290315, 42%), cow (511957, 42%), pig (397065, 44%), dog (477365, 42%), horse (100147648, 42%), chicken (422034, 42%), mouse (58865, 41%), and human (BAB71562.1, 30%). The genomic information may frequently suggest these enzymes are GalE homologues, and we sought distinguishing consensus sequences among these homologues by considering the structural features of T. volcanium l-ThrDH. This approach enabled us to identify a GTTDY motif situated within loop 1 (substrate binding loop) that is strictly conserved among GalE-like l-ThrDH homologues but is not present in GalEs (Fig. 4). This may make the GTTDY motif a benchmark that distinguishes the GalE-like l-ThrDHs from GalEs although further experimental verification should be necessary.