reductase of Thermus thermophilus HB8

Chemico-Biological Interactions 178 (2009) 117–126

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Biochemical and structural characterization of a short-chain dehydrogenase/reductase of Thermus thermophilus HB8 A hyperthermostable aldose-1-dehydrogenase with broad substrate specificity Yukuhiko Asada a,1 , Satoshi Endo b,c,1 , Yukari Inoue b , Hiroaki Mamiya b , Akira Hara b , Naoki Kunishima a , Toshiyuki Matsunaga b,∗ a

RIKEN SPring-8 Center, Harima Institute, 1-1-1 Kouto, Sayo-cho, Sayo-gun, Hyogo 679-5148, Japan Laboratory of Biochemistry, Gifu Pharmaceutical University, Mitahora-higashi, Gifu 502-8585, Japan c United Graduate School of Drug Discovery and Medical Information Sciences, Gifu University, Gifu 501-1193, Japan b

a r t i c l e

i n f o

Article history: Available online 24 September 2008 Keywords: Short-chain dehydrogenase/reductase Aldose 1-dehydrogenase Crystal structure Thermostability Aromatic–aromatic interaction Thermus thermophilus

a b s t r a c t Thermus thermophilus HB8 is a hyperthermophilic bacterium, thriving at environmental temperature near 80 ◦ C. The genomic analysis of this bacterium predicted 18 genes for proteins belonging to the short-chain dehydrogenase/reductases (SDR) superfamily, but their functions remain unknown. A SDR encoded in a gene (TTHA0369) was chosen for functional and structural characterization. Enzymatic assays revealed that the recombinant tetrameric protein has a catalytic activity as NAD+ -dependent aldose 1-dehydroganse, which accepts various aldoses such as d-fucose, d-galactose, d-glucose, l-arabinose, cellobiose and lactose. The enzyme also oxidized non-sugar alicyclic alcohols, and was competitively inhibited by hexestrol, 1,10-phenanthroline, 2,3-benzofuran and indole. The enzyme was stable at pH 2–13 and up to 85 ◦ C. We have determined the crystal structure of the enzyme–NAD+ binary complex at 1.65 Å resolution. The structure provided evidence for the strict coenzyme specificity and broad substrate specificity of the enzyme. Additionally, it has unusual features, aromatic–aromatic interactions among Phe141 and Phe249 in the subunit interface and hydrogen networks around the C-terminal Asp–Gly–Gly sequence at positions 242–244. Stability analysis of the mutant D242N, F141A and F249A enzymes indicated that the two unique structural features contribute to the hyperthermostability of the enzyme. This study demonstrates that aldose 1-dehydrogenase is a member of the SDR superfamily, and provides a novel structural basis of thermostability. © 2008 Elsevier Ireland Ltd. All rights reserved.

1. Introduction The short-chain dehydrogenases/reductases (SDRs) are a large superfamily of NAD(P)(H)-dependent oxidoreductases [1–3] which are distinct from the medium-chain dehydrogenases/reductases (MDR) [4] and aldo-keto reductase (AKR) superfamilies [5]. They are expressed across all phyla, and there are over 15,000 pri-

Abbreviations: SDR, short-chain dehydrogenase/reductase; AKR, aldo-keto reductase; MDR, medium-chain dehydrogenase/reductase; DrSDR, Deinococcus radiodurans SDR family oxidoreductase; TAD, T. thermophilus aldose 1-dehydrogenase; GlcD, glucose 1-dehydrogenase; RMSD, root mean square deviation; HSD, hydroxysteroid dehydrogenase; TaGlcD, Thermoplasma acidophilum d-aldohexose dehydrogenase; BmGlcD, Bacillus megaterium glucose dehydrogenase; CPK, Corey–Pauling–Koltum. ∗ Corresponding author. Tel.: +81 58 237 3931; fax: +81 58 237 5979. E-mail address: [email protected] (T. Matsunaga). 1 Contributed equally to this work and share first authorship. 0009-2797/$ – see front matter © 2008 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.cbi.2008.09.018

mary structures annotated in the various sequence databases. The majority of these proteins have low sequence identity, but share common sequence motifs that define the N-terminal coenzyme binding-site (TGxxxGxG) and the catalytic residues (N-S-Y-K). In humans at least 63 SDR genes exist, where they play physiological roles in the metabolism of steroid hormones, prostaglandins, carbohydrates and retinoids, as well as in the metabolism of xenobiotics, drugs and carcinogens. In other organisms, a huge amount of information on gene sequences encoding SDRs is provided from large-scale genome sequencing projects. However, for a considerable fraction of the predicted gene products, we are far from being assign their functions, particularly because of the large superfamily and low sequence identity among various SDRs with diverse functions. Thermus thermophilus HB8 is an extremely thermophilic bacterium, which can grow at 80–85 ◦ C. A recent genomic analysis of this bacterium has predicted about 2260 genes [6 and http://www.thermus.org/], of which 18 genes encode proteins

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Fig. 1. Sequence alignment of TAD with TaGlcD and D. radiodurans SDR family oxidoreductase (DrSDR). Residues involved in substrate binding and catalysis of TAD and TaGlcD are shown by white letters on a black background, and their coenzyme-binding residues are highlighted against a grey background. Secondary structure elements determined for TAD are indicated above the alignment.

belonging to the SDR superfamily. Most of the predicted T. thermophilus SDRs do not show significant homology against other characterized SDRs of other species, and thus their functions remain unknown. In order to elucidate the functions of T. thermophilus SDRs and structural insight for their extremely high thermostability, we have selected and characterized one SDR encoded in a gene (TTHA0369), because its 256-amino acid sequence is identical to that of a TTC0002 gene product predicted by the genomic analysis of another strain, T. thermophilus HB27 [7] (Fig. 1). It also shows high sequence identity (56%) with a functionally unknown gene product (DrSDR) of Deinococcus radiodurans R1 belonging to the same bacterial clade as T. thermophilus (accession no. Q9RWD9). Here, we report the biochemical and structural characterization of the TTHA0369 gene product. Our work shows that the gene product exhibits NAD+ -dependent dehydrogenase activity for various aldoses and several non-sugar alicyclic alcohols. Based on the substrate specificity for sugars and reaction catalyzed, this enzyme is classified as aldose 1-dehydrogenase (EC 1.1.1.121). The determination of the crystal structure of T. thermophilus aldose 1dehydrogenase (TAD) at 1.65 Å resolution provides the structural basis of the coenzyme specificity, broad substrate specificity and hyperthermostability. 2. Materials and methods 2.1. Protein expression and purification Protein expression and purification using the expression plasmid pET-11a carrying the gene for TAD (residue 1–256) [6] were performed according to the following procedure. Escherichia coli BL21 (DE3) cells were transfected with the expression plasmid and grown at 37 ◦ C in a Luria–Bertani medium containing 50 ␮g/ml ampicillin for 20 h. The cells were harvested by centrifugation (4500 × g for 5 min) at 4 ◦ C, suspended in 20 mM Tris–HCl, pH 8.0 (buffer A), containing 0.5 M NaCl and 5 mM 2-mercaptoethanol, disrupted by sonication, and heated at 70 ◦ C for 10 min. After the heat-denaturized proteins and cell debris were removed by the centrifugation (20,000 × g for 20 min), the sample was desalted on a HiPrep 26/10 desalting column (Amersham Biosciences) and applied to a Super Q Toyopearl 650 M column (Tosoh) that had been equilibrated with buffer A. TAD was eluted with a linear gradient of 0–1.0 M NaCl. The enzyme fraction was desalted, and applied to a Resource Q column (GE Healthcare Biosciences) equilibrated with buffer A. TAD was eluted with a linear gradient of 0.1–0.3 M NaCl. The buffer in the enzyme fraction was replaced with 10 mM sodium

phosphate buffer, pH 7.0, using the HiPrep 26/10 desalting column, and the enzyme sample was applied to a Bio-Scale CHT-20-I column (Bio-Rad) equilibrated with the phosphate buffer. TAD was eluted with a linear gradient of 0.01–0.25 M sodium phosphate buffer, pH 7.0. The enzyme fraction was concentrated by ultrafiltration and loaded onto a HiLoad 16/60 Superdex 200 prep-grade column (GE Healthcare Bio-Science) equilibrated with buffer A containing 0.15 M NaCl. TAD (27-kDa) was identified by Coomassie brilliant blue staining on SDS-PAGE analysis in each step of the purification, which was carried out at room temperature. Finally, the purified TAD was concentrated to 13 mg/ml by ultrafiltration and stored at 4 ◦ C. 2.2. Crystallization and structure determination The Hampton Crystal Screen kit [8] was used to determine the crystallization conditions for TAD. Crystallization was carried out by the oil-microbatch method [9] using Nunc HLA plates (Nalge Nunc International). Each crystallization drop was prepared by mixing 0.5 ␮l precipitant solution and 0.5 ␮l TAD solution (13 mg/ml in 0.2 M NaCl and 20 mM Tris–HCl, pH 8.0). The crystallization drop was overlaid with a 1:1 mixture of silicone and paraffin oils, allowing slow evaporation of water in the drop, and stored at 18 ◦ C. After 1-week incubation, plate-like crystals of the enzyme grew to typical dimensions of 0.20 mm × 0.20 mm × 0.20 mm using the precipitant solution no. 6 of Crystal Screen I (0.2 M magnesium chloride, 30% (w/v) polyethylene glycol 4000 and 0.1 M Tris–HCl, pH 8.5). The crystals were soaked in a cryoprotectant solution no. 6 of Crystal Screen Cryo (0.16 M magnesium chloride, 20% (w/v) glycerol, 24% (w/v) polyethylene glycol 4000 and 0.08 M Tris–HCl, pH 8.5). All Xray diffraction data sets were collected from directly flash-cooled crystals from crystallization drop at −173 ◦ C. The diffraction data for the enzyme were collected using a Rigaku R-AXIS V image-plate detector and synchrotron radiation at beamline BL26B1 of SPring-8, Japan [10]. All the measured diffraction spots were indexed, integrated and scaled using HKL2000 program package [11]. The structure of TAD was solved by the molecular replacement technique [12] with the coordinate set of the ternary complex of Datura stramonium tropinone reductase-II (PDB code: 1IPE) as a search model. Model building of TAD was first performed with ARP/wARP [13] using the warpNtrace automated procedure. Model was built using the graphic program QUANTA (Accelrys, San Diego, CA) and refined using the program CNS version 1.1 [14]. Several cycles of the model building and refinement yielded the final model. The quality of the final structure was evaluated using PROCHECK

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[15]. The analysis of the secondary structure composition was performed with DSSP [16]. Fold-similarity searches were performed with the DALI server [17]. Superposition of protein models was performed using the program LSQKAB [18]. All visualization and image production were prepared using PyMol (DeLano Scientific, San Carlos, CA, USA). The atomic coordinates and structure factor amplitudes of TAD are deposited in the RCSB Protein Data Bank (PDB) with the entry code 2D1Y. 2.3. Enzyme activity assay Dehydrogenase and reductase activities of TAD were assayed by measuring the rate of change in NADH absorbance at 340 nm. The standard reaction mixture for the dehydrogenase activity consisted of 0.1 M potassium phosphate, pH 7.4, 1 mM NAD+ , 0.2 M d-galactose and enzyme, in a total volume of 2.0 ml. The reaction was carried out at 50 ◦ C, otherwise noted. In the assay for the reductase activity, 0.1 mM NADH was used as the coenzyme. The sugars and other non-sugar substrates of the highest purity were obtained from Sigma–Aldrich Chemicals, Nacalai Tesque and Tokyo Kasei Kogyo. The water-insoluble substrates and inhibitors were dissolved in methanol, and added into the reaction mixture, in which the final concentration of methanol was less than 2.5%. One unit (U) of enzyme activity was defined as the amount of enzyme that catalyzes the formation or oxidation of 1 ␮mol of NADH. In the experiments on the effect of pH on the activity and stability, following 0.1 M buffers were employed: KCl–HCl (pH 1–2), glycine–HCl (pH 3), sodium acetate (pH 4–5), potassium phosphate (pH 6–8), glycine–NaOH (pH 9–10), sodium phosphate–NaOH (pH 11), and KCl–NaOH (pH 12–13). The apparent Km and kcat values were determined over a range of five substrate concentrations at a saturating concentration of coenzyme by fitting the initial velocities to the Michaelis–Menten equation. Kinetic studies in the presence of inhibitors were carried out in a similar manner, after IC50 (concentration required for 50% inhibition) values for the inhibitors were determined. The inhibitor constant (Ki ) was calculated by using the appropriate programs of the ENZFITTER (Biosoft, Cambridge, UK). Kinetic constants are expressed as the means of three determinations. The products of d-glucose oxidation were analyzed by the method of Giardina et al. [19]. 2.4. Site-directed mutagenesis Mutagenesis was performed using a QuickChange site-directed mutagenesis kit (Stratagene) and the expression pET11a vector harboring the gene for TAD. The 25–28 mer mutagenic primers were designed to produce the mutant enzymes of F141A, D242N and F249A by replacing the respective codons in the gene with an Ala codon, GCC, or Asn codon, AAC. The complete coding regions of the cDNAs were sequenced with a CEQ2000XL DNA sequencer (Beckman Coulter) to confirm the presence of the desired mutations and to ensure that no other mutation had occurred. The mutant enzymes were expressed in E. coli BL21(DE3), and purified by the anion exchange chromatography and gel filtration as described above. 3. Results and discussion 3.1. Biochemical characteristics of TAD 3.1.1. Purification of recombinant TAD SDS-PAGE analysis of the E. coli cell extract displayed that a 27-kDa protein is overexpressed in the cells transfected with the expression plasmids harboring the TTHA0369 gene (data not shown). The 27-kDa protein was purified from the cell extract

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Table 1 Substrate specificity in the oxidation of carbohydrates. Substrate

Km (mM)

kcat (min−1 )

kcat /Km (min−1 /mM)

Monosaccharides d-Fucose 2-Deoxy-d-galactose d-Galactose 6-Deoxy-d-glucose 2-Deoxy-d-glucose d-Talose l-Arabinose d-Galactosamine d-Glucose d-Glucosamine l-Fucose d-Xylose

5.5 33 78 178 300 131 262 23 580 139 162 841

524 768 495 381 416 181 273 18.7 231 37.3 6.4 25.4

95 23 6.4 2.1 1.4 1.4 1.0 0.81 0.40 0.27 0.04 0.03

Disaccharides Cellobiose Lactose

250 781

346 554

1.4 0.71

by heat treatment (at 70 ◦ C) and consecutive column chromatographic fractionation on the ion-exchange resins, hydroxylapatite and Superdex 200. Sixty-one milligrams of the electrophoretically homogenous protein was obtained from 1 l of the cultured cells, and its N-terminal amino acid sequence of XGLFAG was identical to that deduced from the gene for TAD (Fig. 1), indicating its identity of the purified protein with the gene product. The protein was eluted at a high-molecular weight of approximately 100 kDa on the Superdex 200 gel filtration, suggesting its tetrameric nature. 3.1.2. Substrate specificity profile When various alcoholic compounds were tested as the substrates, we found that TAD exhibits dehydrogenase activity for a variety of aldoses with NAD+ , but not with NADP+ , as the coenzyme (Table 1). The excellent substrate with the lowest Km value and highest catalytic efficiency (kcat /Km ) was d-fucose (6-deoxyd-galactose), followed by 2-deoxy-d-galactose and d-galactose. Activity was found with d-glucose (the C4 epimer of d-galactose) and its deoxy-derivatives, although the catalytic efficiency was lower. Good activity was observed with d-talose (the C2 epimer of d-galactose), but not with its C4 epimer d-mannose, that corresponds to the epimer of d-galactose at C2 and C4. The enzyme also moderately oxidized l-arabinose, an aldopentose with identical configuration to d-galactose at C2–C4, whereas the activity towards d-xylose (the C4 epimer of l-arabinose) was much lower. It therefore appears that the stereo-configuration at C2 and C4 on sugar molecule greatly influences their interaction with the enzyme. In contrast to the above d-aldohexose substrates, l-fucose was hardly oxidized, and its C4 epimer, l-rhamnose, did not serve as the substrate. While TAD oxidized the two aldopentoses, larabinose and d-xylose, no activity was observed with l-xylose (the C3 epimer of l-arabinose), d-ribose (the C3 epimer of d-xylose) and 2-deoxy-d-ribose. The low or no reactivity suggested the importance of the d-galactose-specific configuration at C3 and C5 in the interaction with the substrate-binding residues of the enzyme. Furthermore, TAD did not oxidize ketoses (d-fructose and d-sorbose), sugar alcohols (d-sorbitol, galactitol, inositol, l-arabitol and xylitol), uronic acids (d-galacturonic acid and d-glucuronic acid), and aldonic acids (l-gulonic acid and 2-keto-l-gulonic acid). The substrate specificity indicates that TAD catalyzes the dehydrogenation of the C1-hydroxyl group of pyranose forms of the aldose substrates. This was again supported by the fact that this enzyme oxidized disaccharides with a C1-hydroxyl group (cellobiose and lactose), in contrast to no activity towards non-reducing disaccharides (sucrose and trehalose) and ␣-methyl-d-glucoside. Indeed,

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the oxidized product of d-glucose by TAD was identified with gluconic acid, which was spontaneously formed by rapid hydrolysis of the unstable reaction product, glucono-␦-lactone, as reported for Sulfolobus solfataricus glucose 1-dehydrogenase (GlcD) [19]. In order to further characterize the enzyme, initial rate measurements were performed at 50 ◦ C and pH 7.4, with NAD+ and d-fucose as substrates. The double reciprocal plots of initial velocity versus concentration of d-fucose at fixed levels of NAD+ yielded a series of interacting lines, which were linear over the range of NAD+ concentrations from 1 to 20 ␮M and of d-fucose concentrations from 4 to 20 mM (data not shown). Secondary plots of the slopes and intercepts of the primary plots against the reciprocal of the substrates were linear within the experimental error. The results are consistent with a sequential mechanism [20], and the kinetic constants were calculated from the intercepts and slopes of the secondary plots using the initial rate equation for this kinetic mechanism: v = VAB/(AB + KaB + KbA + KiaKb). They are: the limiting Michaelis constant for NAD+ , Ka = 1.7 ␮M, the limiting Michaelis constant for d-fucose, Kb = 3.2 mM, the dissociation constant for NAD+ , Kia = 11 ␮M, and the extrapolated catalytic constant, V = 570 min−1 . TAD also exhibited dehydrogenase activity towards several non-sugar alicyclic alcohols (Table 2), although not towards aromatic alcohols (benzyl alcohol and catechol), aliphatic alcohols (1-nonanol, 1-decanol, 3-hydroxybutyric acid and 2,3butanediol), and hydroxysteroids (androsterone, testosterone, 20␣/␤-hydroxyprogesterone, cholic acid, cortisol and corticosterone). Alicyclic alcohols fused to an aromatic ring, such as (S)-(+)-1,2,3,4-tetrahydro-1-naphthol (S-tetralol) and (S)-(+)-1indanol (S-indanol), were excellent substrates, showing lower Km and higher kcat values than the values for d-fucose. When two substrates, d-galactose and S-tetralol, were mixed at their Km values, the rate of dehydrogenation by the enzyme was 65% of the sum of the rates obtained with the individual substrates. This result is in agreement with the theory that an enzyme catalyzes two reactions [21]. In the reverse reaction using NADH as the coenzyme, TAD reduced 0.2 M glucono-␦-lactone (0.35 U/mg), but not the sugars described above. The enzyme also exhibited reductase activity towards some non-sugar carbonyl compounds with aromatic ring (Table 2). No significant activity was observed with other ketones (4-nitroacetophenone, camphorquinone, 2,3-hexanedione and diacetyl) and aldehydes (4-nitrobenzaldehyde, pyridine-3aldehyde, 1-nonanal and citral). TAD exhibited strict coenzyme specificity for NADH in the isatin reductase activity, and no reductase activity was detected with NADPH. The Km value for NADH determined in the presence of 0.1 mM isatin was 23 ␮M.

3.1.3. Inhibitor sensitivity The d-galactose dehydrogenase activity of TAD was inhibited by aromatic compounds, such as hexestrol, 1,10-phenanthroline, 2,3-benzofuran, indole, catechol and indazole, and their IC50 values were 0.063, 0.18, 0.20, 0.48, 1.2 and 1.9 mM, respectively. EDTA (1 mM) and SH-reagents, p-chloromercuriphenylsulfonate and pchloromercuribenzoate (0.1 mM), had no effect on the activity. The inhibition patterns of hexestrol, 1,10-phenanthroline, 2,3benzofuran and indole were uncompetitive with respect to NAD+ and competitive with respect to d-galactose (Ki values are 13, 25, 42 and 480 ␮M, respectively). This implies that the aromatic compounds bind to the substrate-binding site of TAD, and also supports its broad substrate specificity for non-sugar alicyclic alcohols with aromatic ring(s). 3.1.4. Effects of pH, temperature and solvents Both d-galactose and S-tetralol dehydrogenase activities of TAD were similarly elevated by increasing the pH from 6.0 to 11.0. When the enzyme (0.1 mg/ml) was incubated at various pH and 50 ◦ C for 30 min, it was stable at pH ranging from 2.0 to 13.0 and even in 0.1 M NaOH. The effect of temperature on the d-galactose dehydrogenase activity of TAD is shown in Fig. 2. Both kcat and Km values were increased by the elevation of temperature, whereas the kcat /Km values were not largely changed. Similar temperaturedependent changes were observed in the kinetic constants for S-tetralol, d-fucose and d-glucose (data not shown). As T. thermophilus HB8 grows at 80–85 ◦ C, TAD was stable up to 85 ◦ C, and gradually inactivated at higher temperature. Approximately 40% of the activity was inactivated by the incubation of the enzyme solution (0.3 mg/ml in 10 mM potassium phosphate, pH 7.0) for 15 min at 95 ◦ C. Thus, TAD may catalyze the dehydrogenation of the sugars and alicyclic alcohols independently of the environmental temperature. Furthermore, the enzyme was stable when it was incubated in 10 mM phosphate buffer containing 50% (v/v) methanol or 60% (v/v) ethanol at 50 ◦ C for 15 min. The stability against highly alkaline pH, high temperature and high solvent conditions are typical features of enzymes that are categorized as extremozymes [22].

Table 2 Substrate specificity for non-sugar compounds. Substrate Oxidation 1-Acenaphthenol S-Tetralol S-Indanol 4-Chromanol 2-Cyclohexen-1-ol cis-benzene dihydrodiol R-Indanol R-Tetralol Reduction Isatin Phenyl-1,2-propanedione ␣-Tetralone

Km (mM)

kcat (min−1 )

kcat /Km (min−1 /mM)

0.14 0.17 0.65 0.25 3.7 27 1.3 2.5

26,000 9,700 8,320 2,420 210 443 10 4.5

1.8 × 105 5.7 × 104 1.3 × 104 3.7 × 103 57 16 7.7 1.8

58,900 1,010 234

4.9 × 106 505 123

0.012 2.0 1.9

Fig. 2. Effect of temperature on dehydrogenase activity of TAD. Km (mM), kcat (min−1 ) and kcat /Km (min−1 mM−1 ) for d-galactose were determined at indicated temperatures.

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3.2. Comparison of TAD with other dehydrogenases for aldoses

Table 3 Summary of data collection and model refinement statistics.

The broad substrate specificity for sugars of TAD is different from relatively narrow substrate specificity of bacterial NAD(P)+ and NAD+ -dependent GlcDs (EC 1.1.1.47 and EC 1.1.1.181, respectively) belonging to the SDR [23,24] or MDR [19,25] superfamily, NAD+ -dependent galactose 1-dehydrogenase (EC 1.1.1.48) [26] and NAD(P)+ -dependent l-arabinose 1-dehydrogenase (EC 1.1.1.46) [27] belonging to the Gfo/Idh/MocA family. The requirement of NAD(H) for the reaction catalyzed by TAD, together with its amino acid sequence, indicates that this enzyme also differs from quinoprotein glucose dehydrogenase (EC 1.1.5.2), which has recently been identified in T. thermophilus HB8 [28]. On the other hand, the specificity for sugars and coenzymes of TAD resembles that of Pseudomonas NAD+ -dependent aldose 1-dehydrogenase (EC 1.1.1.121) [29]. Thus, we conclude that TAD is aldose 1-dehydrogenase. TAD possesses a unique ability to oxidize non-sugar alicyclic alcohols. This ability has not been reported for bacterial dehydrogenases for aldoses, although it is observed for mammalian NADPHdependent lung carbonyl reductases [30,31] in the SDR superfamily and NAD(P)+ -dependent hydroxysteroid dehydrogenases (HSDs) [32,33] belonging to the AKR superfamily. Dehydrogenases for the C1-hydroxyl groups of d-glucose [25,34], d-galactose [35], l-arabinose [27] and d-fucose [36] are involved in the non-phosphorylative Enter-Doudoroff pathway or its alternative metabolic pathways in archaea and several bacteria. The representative pathway of d-glucose is: d-glucose → d-glucono-␦-lactone → d-gluconate → d-2-keto-3deoxygluconate → pyruvate + glyceraldehydes [34]. The present functional analysis of TAD, together with the recent identification of the quinoprotein glucose dehydrogenase [28], suggests that various aldoses incorporated in T. thermophilus HB8 are metabolized through such metabolic pathways as energy sources. In contrast, the physiological significance of the dehydrogenation of the nonsugar alicyclic alcohols by TAD is obscure at present. Since its Km values for the alicyclic alcohols are lower than those for sugars, TAD may act as a NADH-regenerating enzyme by oxidizing these substrates through opportunistic uptake by T. thermophilus, and function to input NADH into the respiratory network through NADH:quinone oxidoreductase, a subunit of respiratory complex I, that was recently identified in this bacterium [37].

Parameter

Determination

Space group Resolution range (Å) Cell dimensions a (Å) b (Å) c (Å) ˇ (◦ )

P2(1) 30.0–1.65 (1.71–1.65) 71.39 95.33 71.41 108.3

No. of observations No. of unique reflections Completeness (%) Rmergea a (%) Rsym (%) Mean Redundancy Wilson B-factor (Å2 ) Average B-factor (Å2 )

377,387 108,794 100.0 (100.0) 6.4 (30.8) 5.9 (25.3) 12.7 (4.3) 3.5 16.8 21.7

Ramachandran geometry Most favored (%) Allowed (%)

90.1 9.9

RMSD from ideal values Bond length (Å) Bond angles (◦ ) Dihedral (◦ ) Improper (◦ )

0.005 1.2 21.1 0.83

R-factor Free R-factor

0.177 0.197

3.3. Crystal structure of TAD 3.3.1. Structure determination The crystal structure of TAD was solved by the molecular replacement method, and the model was refined to an R-factor of 0.176 (free R-factor of 0.194) at 1.65 Å resolution. The refinement statistics are summarized in Table 3. The final model included one NAD+ molecule for each of the four crystallographically independent subunits (Fig. 3), and a total of 827 water molecules per asymmetrical unit. Missing from the model were the N-terminal Met1 and six C-terminal residues (Met250 to Val256). While the residues 2–249 of subunit C were well defined, other subunits contained disordered regions, which composed of residues 193–200 (for subunit A), 192–198 (for subunit B) and 193–199 (for subunit D). 3.3.2. Tertiary structure The subunit of TAD was a single-domain protein having the ␣/␤ doubly wound structure (Fig. 4). A seven-stranded parallel ␤sheet (␤A to ␤G) in the center of the molecule was sandwiched by two arrays of parallel ␣-helices (␣B to ␣G). Two short helices (␣FG1 and ␣FG2) were somewhat separated from the main body of the subunit, although the loop between the two helices was

a Rmerge = hkl j |Ij (hkl) − |/hkl j Ij (hkl), where Ij (hkl) and are the observed intensities of reflections with an index hkl, for the measurement j and for the mean intensity, respectively.

disordered in subunits A, B and D. A C-terminal extension from the structural core of subunit comprised a loop after ␤G to complete the structure. This C-terminal tail contributes to inter-subunit interactions as described later. The upper depression of the subunit of TAD is the putative active site cleft, and the nicotinamide moiety of the bound NAD+ molecule resided deep in the cleft. This, together with the kinetic analyses, indicates that TAD follows an ordered sequential mechanism in which the coenzyme binds first. The folding topology of TAD is quite similar to those of the other enzymes belonging to the SDR family. Indeed, DALI analysis [17] of the subunit C of TAD identified 24 SDRs with Z cores of 36.7–34.0 and RMSD of 1.2–1.9 Å, which include NADPHdependent enzymes such as mouse lung carbonyl reductase (PDB 1CYD [31]) and human l-xylulose reductase (PDB 1PR9 [38]). Among these SDRs, Thermoplasma acidophilum d-aldohexose dehydrogenase (TaGlcD, PDB 2DTX [23]) and Bacillus megaterium glucose dehydrogenase (BmGlcD, PDB 1GOC [39]) are only NAD+ preferring dehydrogenases for aldoses. Other topologically similar NAD+ -dependent SDRs are 7␣-HSD [40], 3␣(20␤)-HSDs [41,42], 3-hydroxybutyrate dehydrogenases [43,44] and oxidoreductases towards specific substrates, such as meso-2,3-butanediol [45], xylitol [46], levodione [47] and (S)-1-phenylethanol [48]. However, TAD did not accept hydroxysteroids, 3-hydroxybutyrate, 2,3-butanediol and xylitol as the substrates, and its sequence identity percentages with these SDRs are low (27–34%). Fig. 1 shows the sequence alignment of TAD with TaGlcD, which exhibits the highest identity (34%) and is similar to TAD in its specificity for some sugars. 3.3.3. Coenzyme-binding mode The bound NAD+ molecule adopts an extended conformation in which the adenine ring is in an anti conformation and the nicoti-

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Fig. 4. Ribbon diagram of the TAD subunit C. Bound NAD+ molecule is shown in CPK representation. The ␣-helices (purple) and ␤-strands (green), as well as the N-terminal Gly2 (N-ter) and C-terminal Phe249 (C-ter), are marked.

one reductase [47], in which the aspartic acid interacts only with the adenine ribose of the coenzyme. Indeed, the Km and Kd values for NAD+ of TAD are lower than those of these SDRs. In addition to the hydrophobic interactions by the other residues, the dual interactions of Asp37 with the adenine nucleoside moiety may contribute to the high affinity for the coenzyme in TAD. Fig. 3. Ribbon representations of the tetramer of TAD viewed along each of the two non-crystallographic two-fold axes. The subunits A–D are shown in green, blue, pink, and yellow, respectively. The molecule of bound NAD+ is shown in Corey–Pauling–Koltum (CPK) representation. (A) View along the R-axis. The ␣G and ␤G of the subunits A and B are labeled. (B) View along the Q-axis. The ␣FG1 and ␣FG2 of the subunit A, and ␣E and ␣F of the subunit D are labeled.

C2 -endo

namide ring in syn. Both ribose rings have the puckering. The conformation of the bound coenzyme is common among most SDRs, in which the pattern of hydrogen bonds with the coenzymes are in general well conserved [49,50], except for the electrostatic environment surrounding the 2 -hydroxyl (or phosphate) group of the adenosine ribose moiety of NAD(H) (or NADP(H)). Most interactions with NAD+ found in TAD (Table 4) are also similar to those in other SDRs, but there were many hydrophobic interactions with the adenine nucleoside moiety of the coenzyme. The adenine ring moiety was accommodated in a hydrophobic pocket surrounded by the side chains of four non-polar residues and Asp37. The sidechain carboxyl group of Asp37 also formed tight hydrogen bonds with the 2 - and 3 -hydroxyl groups of the adenine ribose. The dual interactions between Asp37 and the adenine nucleoside moiety of NAD+ are probably related to the strict NAD(H) specificity of TAD. Asp37 is not conserved in NAD+ -preferring TaGlcD [25] and BmGlcD [39] that are classified as EC 1.1.1.47 because of exhibiting low activity with NADP+ as the coenzyme. Asp37 is conserved in NAD+ -specific SDRs, such as E. coli 7␣-HSD [40], human d-3hydroxybutyrate dehydrogenase type 2 [44], Gluconobacter oxydans xylitol dehydrogenase [46] and Corynebacterium aquatium levodi-

Table 4 Summary of interactions between NAD+ and TAD. NAD+

TAD

Distance (Å)

Nicotinamide nucleoside moiety O7N Ile182 N N7N Thr184 OG1 Tyr149 OH O2 N Lys153 NZ O2 N Lys153 NZ O3 N Asn84 O O3 N

2.7 3.3 2.6 3.1 2.9 2.6

Pyrophosphate moiety O1PN O2PN O1PA O2PA

Thr184 OG1 Ile18 N Arg16 NE Arg16 NH2

2.7 3.1 3.0 2.9

Adenine ribose moiety O2 A O3 A

Asp37 OD1 Asp37 OD2

2.7 2.6

Adenine ring moiety C2Aa C2Aa C2Aa C4Aa C6Aa C8Aa N1A N6A

Asp37 CA Leu38 CG Leu58 CG Ala85 CB Leu58 CB Ile87 CG2 Leu58 N Asp57 OD1

3.8 3.9 3.8 3.9 3.9 3.7 2.9 3.2

a Hydrophobic contact, in which the nearest atom to the adenine ring moiety is selected on behalf of the relevant residue.

Y. Asada et al. / Chemico-Biological Interactions 178 (2009) 117–126

Fig. 5. Active site of the TAD subunit C. (A) The catalytic triad (S136, Y149 and K153), putative substrate-binding residues and NAD+ in the active site, which are shown in ribbon diagram. (B) The electron density maps corresponding to the unidentified compound (blue), catalytic triad and NAD+ in the active site. (C) Superimposition of the ␣FG1, ␣FG2 and the loop between the two helices of subunit B (sky blue) and subunit C (purple). C␣ traces of the regions are shown, except that NAD+ , catalytic triad and the putative substrate-binding residues in the regions are indicated.

3.3.4. Active site residues The catalytic mechanism of the SDRs, involving the S–Y–K catalytic triad, appears to be common among them [3,49]. The serine plays a role in the catalytic step as a stabilizer of the reaction species, the tyrosine is a catalytic residue, and the lysine has dual roles: in orienting the nicotinamide moiety of the coenzyme and in lowering the pKa value of the tyrosine residue. As observed for many other SDRs, TAD possesses the catalytic triad: Ser136, Tyr149, and Lys153 (Fig. 5A). In addition, Filling et al. [51] proposed a new catalytic mechanism that extends the above-mentioned catalytic triad to form a tetrad of N–S–Y–K, of which the asparagine plays a role in maintaining the active-site configuration to build up a proton relay system. The asparagine is also conserved in TAD (Asn108), and its side chain atoms contribute to a bifurcated hydrogen bond with the main chain atoms of Ile87 [OD1(Asn108)—N(Ile87) and ND2(Asn108)—O(Ile87)] located downstream of ␤D, which constitutes the right-side wall of the catalytic cleft (Fig. 4). The main chain carbonyl atom of Asn108 interacted with the side chain amino of Lys153 through a water molecule. Thus, the catalytic mechanism of TAD appears to be similar to that proposed for the other SDRs. As shown in Fig. 3A, the loop between ␣FG1 and ␣FG2, that was disordered in subunits A, B and D, was defined in subunit C of TAD. An electron density for an unidentified compound was found only in the putative active site cleft of subunit C (Fig. 5B), and the C␣ trace of subunit C was slightly moved inside the active site compared to

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those of the other subunits (Fig. 5C). The position of this compound was well superimposed with those of substrates in the structures of TaGlcD [23] and mouse lung carbonyl reductase [31]. The finding suggests that residues in the loop and/or the two helices are involved in the binding of the unidentified ligand in the subunit C model. The ligand-induced conformation of subunit C corresponds to a so-called closed conformation of SDRs [40,49], whereas the disordered loops in the other subunits without this ligand may reflect the open conformations. Fig. 5A depicts thirteen residues that are proximal (within 4 Å) to the electron density map of the unidentified compound. The residues include the catalytic triad and three residues (Val187, Ala190 and Trp204) in ␣FG1 and ␣FG2, which undergo the conformational change by the binding of the unknown compound. TAD and TaGlcD [23] differ from each other in their substrate specificity. TAD efficiently oxidized d-fucose and d-galactose that are poor and inactive substrates, respectively, for TaGlcD, whereas it did not oxidize d-mannose that is the best substrate for TaGlcD. In the structure of TaGlcD, Glu84 (Ala88 in TAD) interacts with the axial C2 hydroxyl group of d-mannose and is crucial for discrimination between d-mannose and its C2 epimers, d-fucose and d-galactose. The interactions of Thr176 (Ala181 in TAD) with both C4 and C6 hydroxyls of d-mannose contribute to the high activity of TaGlcD towards this sugar. The substitutions of the two residues with alanines in TAD may cause its inability to reduce dmannose, and instead lead to its high activity towards d-fucose, which is the C6-deoxy and C4 epimer of d-mannose. The side chain of Ala88 is also distal to the unidentified compound found in the active site of TAD. Among the proposed active-site residues of TAD, Ala190, Trp204, Leu207 and Met245 of TAD are not suggested as the substrate-binding residues of TaGlcD. In addition to Ala181, these non-polar residues in the active site of TAD might be related to its outstanding characteristic, the high activity towards hydrophobic alicyclic compounds with aromatic rings. 3.3.5. Quaternary structure Like many SDRs, TAD was a homotetramer with 222 point group symmetry resulting in a rectangular block of approximate dimensions of 70 Å × 70 Å × 60 Å (Fig. 3A). The three mutually perpendicular two-fold axes in the tetrameric SDRs are conventionally designated P, Q, and R [41]. The most extensive inter-subunit interactions were found between the Q-axis related subunits. The Q-axis interface comprised two ␣E and two ␣F helices (Fig. 3B). The ␣E and ␣F helices belonging to one subunit were in contact with the ␣E and ␣F helices belonging to the other subunit, respectively. While no interaction, other than two hydrogen bonds between Asn150 and Asn158, was observed in the ␣F–␣F interface, many hydrophobic interactions existed in the ␣E–␣E interface: The hydrophobic side chains of Leu98, Met105, Trp101, Leu109 and Met113 on the helices faced each other. In addition, there were two hydrogen-bonding interactions between the side chains of Trp101 and Thr110, and those of Arg102 and Glu106 in this interface. The guanidino group of Arg102 also formed a salt bridge with the side chain carboxyl group of Glu61 in ␣D belonging to the other subunit. Such hydrogen bond/salt bridge network between Arg102 in ␣E of one subunit and the residues in ␣D and ␣E of the other subunit was not observed in the other SDRs that are topologically similar to TAD in the DALI search. Interactions between P-axis related subunits are not so extensive as compared with those between the Q-axis related subunits. Similarly to the Q-axis interface, the P-axis interface can be described in terms of the ␣G–␣G and ␤G–␤G interfaces (Fig. 3A). In the ␣G–␣G interface the side chains from the symmetry-related Phe225 were stacked and the side-chain carboxyl of Glu218 was hydrogen bonded to the side-chain hydroxyl of Ser232 of the other

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Fig. 7. Effect of mutation on thermostability of TAD. Solutions (0.3 mg/ml in 10 mM phosphate, pH 7.0) of the wild type (), F141A (䊉), F249A () and D242N () enzymes were incubated at indicated temperatures for 15 min, and then the remaining enzyme activity was assayed. Fig. 6. Intra-molecular and inter-subunit interactions in the C-terminal tail of TAD. (A) Close-up of the hydrophobic interactions of Phe141 and Phe249 from the subunit A (green), subunit B (blue), subunit C (pink) and subunit D (yellow). (B) Close-up view of the C-terminal residues (blue) located downstream of ␤G of the subunit B. Phe141 and Phe249 (yellow) of the subunit D are also depicted. Hydrogen bonds are illustrated as black dotted lines and distances are given in Å. (C) Alignment of the C-terminal sequences of TAD, human d-3-hydroxybutyrate dehydrogenase (DHRS6), TaGlcD, BmGlcD, Streptomyces 3␣,20␤-HSD (HSD), Mycobacterium 3␣,20␤-HSD (Rv2002), meso-2,3-butanediol dehydrogenase (mBDH), levodione reductase (LVR), S-1-phenylethanol dehydrogenase (PED), and human l-xylulose reductase (hXR). Conserved D–G–G sequence located downstream of ␤G (underlined) is stressed, and the residues interacted in the R-axis interfaces are shown in red.

subunit. The hydrophobic side-chains of Leu239 on the ␤G helices faced each other. In the R-axis interface of TAD, there were two hydrophobic cores where no water molecule was found. Each core is formed by four phenyl rings from the symmetry-related Phe141 and Phe249 (Fig. 6A). In the pairs of Phe141(A)–Phe249(C), Phe141(A)–Phe141(C) and Phe249 (A)–Phe249(C) in the interface of the two subunits, the phenyl ring centroids were separated by distances of 4.5–6.0 Å and their dihedral angles were within 90◦ , being in agreement with aromatic–aromatic interactions of protein stabilization [52]. The interactions were also maintained by hydrogen bonds between the main-chain atoms of Ala142 (subunit A or B) and Ala247 (subunit C or D). Such aromatic–aromatic interactions were not observed in the structures of all SDRs found by the DALI search. In the crystal structures of mouse lung carbonyl reductase [31] and human l-xylulose reductase [38], the R-axis interface involves one prominent ionic interaction between the C-terminal carboxylate group belonging to one subunit and the guanidino group of Arg203 belonging to the other subunit. In the cases of bacterial TaGlcD [23], BmGlcD [39], S-1-phenylethanol dehydrogenase [48] and 3␣,20␤-HSDs [41,42], the R-axis interfaces are created by several hydrogen bonds and/or van der Waals contacts. It should be noted that the C-terminal tail of TAD protrudes from the main body of the subunit (Fig. 6A). Fig. 6B is a magnification of the C-terminal tail of subunit A, where the residues from Asp242 to Ala247, located just downstream of ␤G, formed a network of hydrogen bonds. It is interesting to note that the D–G–G sequence of the protruded region is mostly conserved in human l-xylulose reductase and the above bacterial SDRs (Fig. 6C). In

human l-xylulose reductase, the role of the residue at position 242 in preventing cold inactivation is demonstrated by site-directed mutagenesis [53]. This sequence is also highly conserved in bacterial and mammalian d-3-hydroxybutyrate dehydrogenases [43,44] and several other SDRs. Indeed, similar hydrogen bond networks around the D–G–G sequence were observed in the structures of the two d-3-hydroxybutyrate dehydrogenases, TaGlcD [23], BmGlcD [39], 3␣,20␤-HSDs [41,42], meso-2,3-butanediol dehydrogenase [45], levodione reductase [47] and S-1-phenylethanol dehydrogenase [48], where the residues in the downstream C-terminal tails are involved in interactions in the R-axis interfaces. These observations suggest that the D–G–G sequence located behind ␤G plays a role in proper orientation of the C-terminal tail for subunit interaction in the above tetrameric SDRs as well as TAD. 3.3.6. Structural basis of hyperthermostability Inter-subunit interactions are potential major stabilization mechanism in many oligomeric and hyperthermophilic proteins [54], including dimeric 3-isopropylmalate dehydrogenase [55], tetrameric homoisocitrate dehydrogenase [56] and tetrameric aldolase [57] of T. thermophilus. In these T. thermophilus enzymes, hydrophobic interactions are suggested or demonstrated to be involved in the thermostable oligomer formation, but the unique aromatic–aromatic interactions found in the present TAD structure have not been reported. To verify the contribution of the aromatic–aromatic interactions and D–G–G sequence to the thermostability of TAD, we prepared the mutant TADs, F141A, F249A and D242N. As shown in Fig. 7, the mutations resulted in decrease in thermostability of TAD without dissociation into dimer or monomer, which were confirmed by the molecular weight analysis of the mutant enzymes by gel-filtration. The large decrease in thermostability by the D242N mutation compared to the other mutations suggests that the C-terminal D–G–G sequence is an important structural factor in TAD. Acknowledgments This work (HTPF 00160) was supported by the National Project on Protein Structural and Functional Analyses funded by the MEXT of Japan.

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