Structural basis for stereo-specific catalysis in NAD + -dependent (R )-2-hydroxyglutarate dehydrogenase from Acidaminococcus fermentans Berta M. Martins 1, *, Sandra Macedo-Ribeiro 2 , Julia Bresser 3 , Wolfgang Buckel 3 and Albrecht Messerschmidt 1 1 MPI Biochemie, Strukturforschung, Martinsried, Germany 2 Centro de Neurocie ˆ ncias e Biologia Celular, Coimbra, Portugal 3 Laboratorium Mikrobiologie, FB Biologie, Philipps-Universita ¨ t, Marburg, Germany The physiological role of NAD + -dependent (R)-2- hydroxyglutarate dehydrogenase (HGDH, E.C. 1.1.1 ) is the reduction of 2-oxoglutarate (pK eq ¼ 11.8) to (R)-2-hydroxyglutarate coupled with the oxidation of NADH to NAD + (Scheme 1). The reaction equilibrium constant (K eq ¼ 1.47 · 10 )12 M ) lies on the side of (R)-2-hydroxyglutarate production [1]. The optimum pH is 8.0 (specific activity of 4800 UÆmg )1 ) with a K m value of 134 l M for 2-oxoglutarate (at 100 l M NADH) [2]. In Acidaminococcus fermentans, a nonpathogenic Gram-negative bacterium (order Clostridiales, family Acidaminococcaceae) commonly found in the digestive tract of mammals [3], HGDH catalyses the second step of the hydroxyglutarate pathway in glutamate fer- mentation, the prominent energy metabolism in the microorganism [4]. During catalysis, the 4Re-hydrogen Keywords D -2-hydroxyacid dehydrogenase; NAD + - dependent hydride transfer; (R)-2- hydroxyglutarate; substrate recognition ⁄ stereospecificity; phase combination Correspondence B. M. Martins or A. Messerschmidt, MPI Biochemie, Strukturforschung, 82152 Martinsried, Germany Fax: +49 89 85783516 Tel: +49 89 85782669 E-mails: martins@biochem.mpg.de; messersc@biochem.mpg.de Website: http://www.biochem.mpg.de/xray *Present address Laboratorium Proteinkristallographie, Univer- sita ¨ t Bayreuth, 95440 Bayreuth, Germany (Received 27 July 2004, revised 29 September 2004, accepted 29 September 2004) doi:10.1111/j.1432-1033.2004.04417.x NAD + -dependent (R)-2-hydroxyglutarate dehydrogenase (HGDH) cata- lyses the reduction of 2-oxoglutarate to (R )-2-hydroxyglutarate and belongs to the D -2-hydroxyacid NAD + -dependent dehydrogenase ( D -2-hydroxyacid dehydrogenase) protein family. Its crystal structure was determined by phase combination to 1.98 A ˚ resolution. Structure–function relationships obtained by the comparison of HGDH with other members of the D -2- hydroxyacid dehydrogenase family give a chemically satisfying view of the substrate stereoselectivity and catalytic requirements for the hydride trans- fer reaction. A model for substrate recognition and turnover is discussed. The HGDH active site architecture is structurally optimized to recognize and bind the negatively charged substrate 2-oxoglutarate. The structural position of the side chain of Arg52, and its counterparts in other family members, strongly correlates with substrate specificity towards substitutions at the C3 atom (linear or branched substrates). Arg235 interacts with the substrate’s a-carboxylate and carbonyl groups, having a dual role in both substrate binding and activation, and the c-carboxylate group can dock at an arginine cluster. The proton-relay system built up by Glu264 and His297 permits His297 to act as acid–base catalyst and the 4Re-hydrogen from NADH is transferred as hydride to the carbonyl group Si-face lead- ing to the formation of the correct enantiomer (R)-2-hydroxyglutarate. Abbreviations HGDH, NAD + -dependent (R)-2-hydroxyglutarate dehydrogenase; FDH, NAD + -dependent formate dehydrogenase; GDH, NAD + -dependent D -glycerate dehydrogenase; PGDH, NAD + -dependent D -3-phosphoglycerate dehydrogenase; LDH, NAD + -dependent D -lactate dehydrogenase; HicDH, NAD + -dependent D -2-hydroxyisocaproate dehydrogenase. FEBS Journal 272 (2005) 269–281 ª 2004 FEBS 269 (Prelog–Helmchen Re ⁄ Si-system [5]), at the C4N atom of the nicotinamide moiety of the reduced coenzyme (NADH), is transferred as hydride [6], making HGDH an ‘A-side’ specific dehydrogenase [7,8]. HGDH is a member of the D -2-hydroxyacid dehy- drogenase protein family [9]. Other members structur- ally characterized are formate dehydrogenase ([10,11], Protein Data Bank (PDB) codes 2NAC and 2NAD for apo- and holoFDH, respectively), D -glycerate dehydro- genase ([12], PDB code 1GDH for apoGDH), D -3-phos- phoglycerate dehydrogenase ([13], PDB code 1PSD for holoPGDH), D -lactate dehydrogenase (PDB code 2DLD for the ternary complex of LDH with oxamate; [14]; PDB code 1J49 for h oloLDH [15]) and D -2-hydroxy- isocaproate dehydrogenase ([16], PDB code 1DXY for the ternary complex of HicDH with 2-oxoisocaproate). This protein family has considerable biotechnological potential in the chiral synthesis of both organic com- pounds and nonproteogenic amino acids for pharma- ceutical and medical applications (e.g. used as a diagnostic reagent to monitor the serum levels of amino acids that accumulate in a range of metabolic diseases) [17,18]. D -2-Hydroxyacid dehydrogenases are structurally folded into two domains joined by a hinge and separ- ated by a deep cleft containing the active site. The hinge region mediates domain movements involved in enzymatic catalysis (examples for such an enzymatic reaction are given in [19] and references within). Whereas the smaller domain referred to as the catalytic or substrate-binding domain can be quite variable in structure (as expected from the diverse substrate specif- icity), the larger domain designated the nucleotide- binding domain displays a well-preserved fold indicating its evolutionary structural conservation (as expected from its role in coenzyme binding). The same protein overall fold (structural variant of the bab Rossmann fold [20]), is displayed by members of the transcriptional regulators protein family (C-terminal binding protein, CtBP [21]). The key catalytic residues have been identified by biochemical [22–27] and structural analyses ([11– 13,15]). A conserved histidine-glutamate pair functions as a proton-relay system. An arginine lining the active site has been implicated in recognition and binding of the substrate by a salt bridge with the carboxyl group. The coenzyme binding mode, in a ‘boomerang’ confor- mation with the carboxamide of the nicotinamide moi- ety anti to the ribose moiety has been characterized in the crystal structures of holoFDH [11], holoPGDH [13], the ternary complex of HicDH [16] and the holo- and ternary complex of LDH ([15], PDB code 2DLD). This orientation directs the Re-hydrogen (pro-R hydro- gen) of the nicotinamide ring towards the substrate to be reduced. This explains the pro-R (A-side) specificity displayed by these enzymes [28]. However, it remains to be clarified how the protein backbone controls the substrate stereorecognition for each member of the protein family as their substrates range from the small formate (nonchiral) up to the more structurally elabor- ated and larger molecules 2-hydroxyisocaproate or 2-oxoglutarate. Here we present the crystal structure of apoHGDH from A. fermentans determined at a resolution of 1.98 A ˚ . The structural analysis of HGDH together with the comparison with other structurally characterized members of the D -2-hydroxyacid dehy- drogenase protein family further contributes to clarify the molecular mechanisms of substrate stereospecificity and its catalytic turnover. Results and Discussion Structure determination by phase combination and model refinement Unsuccessful approaches HGDH crystallised in the presence of NAD + , 2-oxo- glutarate or oxalylglycine showed no corresponding electron density. An increase in the concentration of these compounds resulted in the growth of partial merohedral twin crystals with cell dimensions of approximately a ¼ b ¼ 64 A ˚ , c ¼ 170 A ˚ , a ¼ b ¼ 90° and c ¼ 120° (twin fraction of 0.42 ± 0.01; partial Scheme 1. The hydroxyglutarate pathway of glutamate fermenta- tion in A. fermentans. The reaction catalysed by HGDH is box- marked, as well as the final products of the metabolic pathway. ETF, electron transferring flavoprotein. NAD + -dependent (R)-2-hydroxyglutarate dehydrogenase B. M. Martins et al. 270 FEBS Journal 272 (2005) 269–281 ª 2004 FEBS merohedral twinning test at http://www.doe-mbi.ucla. edu ⁄ Services ⁄ Twinning ⁄ ). All attempts to interpret and use the data sets failed. Due to the amino acid sequence identity of roughly 30% between HGDH and other members of the D -2-hydroxyacid dehydrogenase protein family (Fig. 1), Patterson search techniques were tried. The best result obtained with AMoRe [29] was for space group P6 1 22 (CC of 31.5 and R-factor of 54.7%). However, the model building was hindered by the poor quality of the calculated phases. A second approach was the use of heavy atom derivatives, and a complete MAD dataset was collected at the Ta L-III edge to a resolution of 2.79 A ˚ . The Ta 6 Br 12 clusters were located in a crystallographic special position (along the z-axis), which hindered the determination of the crystal structure using the phases gained from the MAD experiment alone. Successful methodology The breakthrough to determine the crystal structure was the use of phase combination (see Materials and methods for details). The resulting electron density map was of excellent quality and it was possible to assign the complete amino acid sequence. The model was refined to 1.98 A ˚ resolution with R cryst of 0.177, R free of 0.225 and good stereochemistry (Table 1). The refined model includes the complete polypeptide chain (Met1–Lys331) and 396 solvent molecules. Disordered side chains (no clear electron density) of residues at the surface of the protein were modelled according to the more stable conformer. The electron density for the cis- peptide bond between residues Tyr289 and Pro290 is clearly defined. Statistics for data processing, phasing and structure refinement are shown in Table 1. The overall topology of A. fermentans HGDH The HGDH homodimer is an elongated ellipsoid with twofold symmetry and approximate dimensions of 90 · 52 · 49 A ˚ 3 (Fig. 2A). This dimer arrangement is conserved among all structurally characterized mem- bers of the D -2-hydroxyacid dehydrogenase family, namely FDH [10,11], GDH [12], PGDH [13], LDH Fig. 1. Structure-based amino acid sequence alignment of HGDH and other members of the D -2-hydroxyacid dehydrogenase protein family. HGDH from A. fermentans (this study); LDH from Lactobacillus bulgaricus (PDB code 1J49 [15], sequence identity 36.5% and similarity 53.8%; 39.0 and 2.1 A ˚ for Z-score and rmsd values, respectively); HicDH from Lactobacillus casei (PDB code 1DXY [16], sequence identity 33.3% and similarity 56.0%; 37.6 and 2.5 A ˚ for Z-score and rmsd values, respectively); LDH from Lactobacillus helveticus (PDB code 2DLD, sequence identity 38.3% and similarity 54.7%; 35.0 and 2.5 A ˚ for Z-score and rmsd values, respectively); FDH from Pseudomonas sp. 101 (PDB code 2NAD [11], sequence identity 21.6% and similarity 34.8%; 28.1 and 3.8 A ˚ for Z-score and rmsd values, respectively); and PGDH from Escherichia coli (PDB code 1PSD [13], sequence identity 19.9% and similarity 30.7%; 31.6 and 2.3 A ˚ for Z-score and rmsd values, respectively). The secondary structure elements of HGDH are displayed by rods for helices and arrows for strands and coloured blue and green according to the respective substrate- and nucleotide-binding domains. The asterisks indicate the catalytic triad residues, Arg235, Glu264 and His297 (numbers for HGDH). Lowercase letters indicate amino acid sequence insertions. The residues discussed below are col- oured red for arginine, blue for glutamate and aspartate, green for histidine, cyan for polar, orange for aromatic and aliphatic side chains, brown for Thr77–Gly79 and dark red for Gly153-Asp176. The alignment was produced using the tool CLUSTALW from EMBL-EBI (http:// www.ebi.ac.uk), and the Z-score and rmsd values were obtained with the tool DALI from EMBL-EBI (http://www.ebi.ac.uk). B. M. Martins et al. NAD + -dependent (R)-2-hydroxyglutarate dehydrogenase FEBS Journal 272 (2005) 269–281 ª 2004 FEBS 271 (PDB code 2DLD, [14,15]), and HicDH [16]. This indicates that the quaternary structure of HGDH rep- resents the physiological dimer in solution [4] as also found for the other family members (FDH [30]; GDH [31]; PGDH, where the dimer is part of the functional tetramer [32]; LDH [22]; and HicDH [33]). The dimer interface, built up mainly by helix aA from the nucleo- tide-binding domain, has a hydrophobic character with few salt bridges and solvent molecules involved. Helix aA lies diagonal across the interface and together with aC build up the reverse side while helices aB and aF (one from each monomer) pack together at the front of the interface (in the orientation displayed in Fig. 2A). Each monomer has the shape of a dumb-bell and consists of two compact domains separated by a cleft that is approximately 18 · 15 A ˚ 2 wide and 12 A ˚ deep (Fig. 2B). Both domains are structural variants of the bab Rossmann fold [20] and their related topology, with several a-helices packed against an open twisted b-sheet, can be seen from the rmsd of 1.8 A ˚ for the respective superimposed C a atoms. The smaller domain (upper domain in Fig. 2B; resi- dues 1–101 and 302–331), composed of a five-stranded parallel b-sheet flanked by five helices, is designated the substrate-binding domain. The larger domain (lower domain in Fig. 2B; residues 102–301), compri- sing a seven-stranded parallel b-sheet flanked by seven helices, contains the consensus nucleotide-binding sequence motif GXGX 2 GX 17 D [GXGX 2 GX 17 D(153– 176) in HGDH; Fig. 1] and is therefore referred as the nucleotide-binding domain. The strictly conserved Asp176 is the key residue in the recognition and bind- ing of the cofactor NAD + (instead of NADP + [34]). The two domains are connected through a hinge region (residues 100–102 and 299–302, coloured green in Fig. 2B) that mediates the interdomain flexibility required for efficient substrate binding and catalysis (closure of the interdomain cleft and consequent shielding of the active site from solvent [35]). The superposition of HGDH with other structurally characterized D -2-hydroxyacid dehydrogenases clearly shows that, as expected by the absence of bound coen- zyme or substrate, HGDH displays an open conforma- tion (Fig. 3). The closure of the interdomain cleft results from the rotation of the substrate-binding domain (as a rigid body) towards the nucleotide-binding domain from the position in the apo (open conformation, Fig. 3B) to that found in the holo or ternary complexes (closed conformation, Fig. 3A for holoFDH and holoLDH) [36]. In agreement with this, it is the substrate-binding domain that displays the highest conformational vari- ability (Fig. 3). Interestingly, the ternary complexes of HicDH [16] and LDH show the enzyme in the open conformation with both coenzyme and substrate (2-oxo- isocaproate partially superimposed with a sulfate ion Table 1. Statistics on diffraction data, phasing and structure refinement. Mean I ⁄ r is the mean signal to noise ratio, where I is the intensity of the reflection and r is the estimated error in the measurement; R sym ¼ S h S i |I h,i –<I h >|⁄S h S i |I h,I |; where I is the integrated intensity of reflection h having i observations and < I h > is the mean intensity of reflection h over multiple recording; R cryst ¼ S||F o |–|F c || ⁄S|F o |; where F o and F c are the observed and calculated structure factors. R free is calculated for 5% randomly chosen reflections. Measure Data collection Native MAD-Ta X-ray wavelength (A ˚ ) 1.0500 1.2545 1.2555 1.0500 Resolution (A ˚ ) 20.00–1.98 20.00–2.79 20.00–2.79 20.00–2.76 Total ⁄ unique observations 533627 ⁄ 30959 71420 ⁄ 19159 71581 ⁄ 19152 60515 ⁄ 19338 Completeness (%) a 98.8 (93.9) 98.4 (91.9) 98.4 (91.8) 99.0 (97.8) Mean I ⁄ r a 28.17 (6.84) 18.34 (6.84) 18.43 (6.93) 16.66 (6.80) R sym (%) a 5.3 (31.6) 5.3 (20.6) 5.5 (19.1) 5.4 (17.7) FOM (20.00–5.0 A ˚ ) 0.53 (after SHARP) Structure refinement Resolution range (A ˚ ) ⁄ R cryst ⁄ R free 20.00–1.98 ⁄ 0.177 ⁄ 0.225 Protein atoms; B factor (A ˚ 2 ) ⁄ Solv. molecules; B factor (A ˚ 2 ) 2564 (24.8) ⁄ 396 (37.6) Rmsd geometry bond length (A ˚ ) ⁄ Bond angle (°) 0.023 ⁄ 1.697 Cruickshanks DPI coordinate error 0.145 Ramachandran plot (%); favored, allowed regions 90.8, 9.2 a Numbers in parentheses represent statistics in the highest resolution shell. NAD + -dependent (R)-2-hydroxyglutarate dehydrogenase B. M. Martins et al. 272 FEBS Journal 272 (2005) 269–281 ª 2004 FEBS in HicDH) or inhibitor (oxamate in LDH) bound at the active site. Additionally the holoLDH monomer in the open conformation displays the bound coenzyme (with partial occupancy) [15]. Active site architecture The active site is located at the bottom of the inter- domain cleft at approximately 12 A ˚ below the surface of the protein with the catalytic triad Arg235, Glu264 and His297 facing into the cleft (Fig. 4, and Fig. 5A for a closer view). The residues lining the active site are all well defined except for Arg235, which displays residual electron density for two possible conforma- tions, one pointing towards His297 (used in the struc- tural analysis) and another pointing towards Asp81. The proton-relay system built up by the histidine- glutamate dyad (His297-Glu264 in HGDH) is con- served in all D -2-hydroxyacid dehydrogenases with the exception of FDH (His-Gln dyad [11]). Both residues are involved in a hydrogen bonding interaction where the negative charge of the glutamate stabilizes the protonated state of the histidine (positively charged). Glu264 shifts the pK a of His297 to a higher value, enabling it to act as an acid–base catalyst at the pH value required for the proton transfer [23]. Apart from participating in the proton transfer reaction, histidine forms a hydrogen bond with the carbonyl group of the substrate (or hydroxyl group in the reverse reaction) and stabilizes the reaction transition state [24]. The third residue of the catalytic triad is Arg235. Its positive guanidinium moiety plays an essential role in the polarization of the substrate’s carbonyl group, increasing its susceptibility for a nucleophilic attack [37]. Additionally, this residue is involved in binding the substrate’s a-carboxylate group [14,16] and in the activation of the coenzyme [11]. A positively charged patch at the top right side of the active site (orientation shown in Figs 4 and 5B) is formed by Arg9, Arg52 and Arg76. In contrast to the well conserved Arg9 and Arg76, residues in the D -2- hydroxyacid dehydrogenase family corresponding to Fig. 2. Structural arrangement of HGDH. (A) Ribbon diagram of the HGDH dimer. The substrate- and nucleotide-binding domains are coloured dark blue and orange for one monomer and light blue and green for the other monomer. The two nucleotide-binding domains build the central core of the dimer, whereas the two substrate-binding domains are at opposite sides. The helices involved in the dimer interface are labelled (asterisks for helices from the second monomer). (B) Topology of the HGDH monomer. The secondary structure is coloured in red for a-and3 10 -helices and blue for b-strands and the hinge region is coloured in green. The catalytic triad Arg235, Glu264 and His292 is represented in sticks and coloured gold for carbon, blue for nitrogen and red for oxygen. B. M. Martins et al. NAD + -dependent (R)-2-hydroxyglutarate dehydrogenase FEBS Journal 272 (2005) 269–281 ª 2004 FEBS 273 Arg52 are extremely variable (Fig. 1). LDHs have a tyrosine (Tyr53), HicDH displays a leucine (Leu51) and FDH has a glutamine (Gln96). This amino acid variability was proposed to play a crucial role in sub- strate specificity [13,16]. Thus the presence of a bulky side chain such as tyrosine or of a positively charged residue such as arginine would control the recognition of the substrate, which in HGDH (Arg52) would have a positive selection effect for the negatively charged substrate 2-oxoglutarate. The arginine cluster is flanked by two patches. A hydrophobic one formed by Ile15, Ala305, Val306 and Met309, and a polar patch Fig. 3. Stereo representation of the super- position of apoHGDH with members of the 2-hydroxyacid dehydrogenase protein family. Orientation as for Fig. 2B. (A) Superposition of apoHGDH (orange) with holoFDH (blue) [11] and holoLDH (lime green) [15]. Both holoFDH and holoLDH display a closed inter- domain cleft. (B) Superposition of apoHGDH (orange) with holoPGDH (lime green) [13], LDH in complex with oxamate (blue) (PDB code 2DLD) and HicDH in complex with 2-oxoisocaproate (grey) [16]. All enzymes display an opened interdomain cleft. Fig. 4. Stereo representation of the interdo- main cleft architecture with the active site pocket. The substrate- and nucleotide-bind- ing domains are coloured blue and green, respectively. Residues are represented by sticks and coloured gold for carbon, blue for nitrogen, red for oxygen and yellow for sul- fur. Phe140* is contributed by the nucleo- tide-binding domain (coloured dark red) from the second monomer. NAD + -dependent (R)-2-hydroxyglutarate dehydrogenase B. M. Martins et al. 274 FEBS Journal 272 (2005) 269–281 ª 2004 FEBS composed of Glu12 (hydrogen-bonded to Arg52), Tyr33 and Asn54. Residues Tyr301 and Phe140* (from the second monomer), located below the arginine cluster (orientation shown in Fig. 4) protect that side of the interdomain cleft from solvent. This suggests that these bulky ⁄ hydrophobic side chains contribute to delineate the architecture of the active site pocket and the consequent modulation of 2-oxoglutarate recogni- tion and binding (through its c-carboxylate group) by the arginine cluster. Similar contributions from the sec- ond monomer are observed in PGDH (with phenyl- alanine [13]) and LDH and HicDH (with isoleucine [15]). On the left topside of the active site (orientation shown in Figs 4 and 5A,B) Thr77, Asp81 and the main chain contributed by Ala78 and Gly79 build up a polar pocket. Structural studies of FDH [11], LDH [14] and HicDH [16] have indicated that this structural motif (Thr77–Gly79 in HGDH) has a functional role in binding the substrate’s a-carboxylate group. Another polar patch formed by the hydrogen-bonded Tyr101 and Ser300 (at the rear of the cleft wall as dis- played in the orientation of Figs 4 and 5A) also contri- butes to the orientation of the substrate within the active site. Tyr101 is substituted by an asparagine in FDH [11], a serine in PGDH [13] and a valine in GDH [12], and Ser300 is exchanged by a phenylalanine in LDH [14], tyrosine in HicDH [16], glycine in PGDH [13] and FDH [11], and serine in GDH [12]. This vari- ability correlates with the dimensions of the active site pocket and thus with the electrostatic properties of the respective substrate: bulky or hydrophobic side chains for aliphatic substrates as in HicDH [16] and LDH [14], and polar side chains for hydrophilic substrates as in PGDH [13] and HGDH (this study). The lower wall of the cleft below the active site dis- plays a hydrophobic character with residues Ile106 and Ile157 at the back of the wall and a mixed patch with Val260 and Asp259 at the front part (orientation shown in Fig. 4). These residues are well conserved (Fig. 1), and together with the aliphatic side chains from Pro103 and Ala107 have been proposed to play a structural role in the higher affinity for NADH than for NAD + (as observed for L -2-hydroxyacid dehydrogenases [38]) and the correct orientation of the coenzyme’s nicotinamide moiety [16]. By packing against the B-side of the pyridinium ring the enzyme assures that only the Re-hydrogen (on the A-side) is exposed for catalysis. Additionally to its interaction with the carboxamide moiety [11], Asp259 is involved in a hydrogen-bonding network with the peptide back- bone of the loop containing Ser300. This structural feature, conserved among other members of the pro- tein family, supports the proposed role of Ser300 and Tyr101 in substrate specificity. All of the critical residues are conserved among the homologous HGDH from Fusobacterium nucleatum A B C Fig. 5. Structural basis for substrate stereo-specificity catalysis in HGDH. (A) Stereo representation of the HGDH active site pocket with modelled NAD + and 2-oxoglutarate (based on the superposi- tion of the available holo and ternary complex structures from the D -2-hydroxyacid dehydrogenases family members). Atom colour code as for Fig. 4 except for NAD + (grey with the carboxamide oxy- gen and nitrogen atoms in red and blue, respectively). The hydro- gen bonding interactions between Glu264 and His297, Ser300 and Tyr101 and Glu12 and Arg52 are indicated by dashed lines, as well as possible hydrogen bonding interactions between 2-oxoglutarte and active site residues. (B) Stereo representation of the solid sur- face ⁄ electrostatic potential representation from the active site pocket of HGDH (colour code as in panel A with 2-oxoglutarate in grey for carbons and red for oxygen atoms). (C) Proposed catalytic mechanism of HGDH. The substrate is positioned within the active site pocket by Arg235, the polar patches and the arginine cluster (panel B), and its Si-face directed towards the A-side of the coen- zyme (docking site omitted for simplicity). The carbonyl group is polarized by Arg235 and the relay charge system (Glu264 orients the imidazole ring of the positively charged His297 to optimize pro- ductive interaction with the substrate), and hydride transfer can therefore occur. B. M. Martins et al. NAD + -dependent (R)-2-hydroxyglutarate dehydrogenase FEBS Journal 272 (2005) 269–281 ª 2004 FEBS 275 Fig. 6. Structural comparison of HGDH with LDH (PDB code 1J49 [15]), and HicDH and implications for substrate specificity. Orien- tation as in Fig. 4. (A) Stereo representation of the solid surface ⁄ electrostatic potential representation from the active site pocket of LDH (holo form in closed conformation). Positively and negatively charged regions are coloured blue and red, respectively. Atom colour code as for Fig. 4 except for NAD + (grey with the carboxamide oxygen and nitrogen atoms in red and blue, respect- ively) (B) Stereo display of the superposition of the active site for HGDH (colour code as for Fig. 4) and LDH (red with NAD + coloured like in panel A and the sulfate ion coloured red for oxygen and yellow for sulfur). (C) Stereo representation of the solid sur- face ⁄ electrostatic potential representation from the active site pocket of HicDH (tern- ary complex in open conformation; colour code as for panel A). Atom colour code as for panel A and 2-oxoisocaproate in grey for carbons and red for oxygen atoms. (D) Ste- reo display of the superposition of the active site for HGDH (colour code as for Fig 4 and 2-oxoglutarate depicted) and HicDH (red with NAD + and 2-oxoisocaproate coloured like in panel C). NAD + -dependent (R)-2-hydroxyglutarate dehydrogenase B. M. Martins et al. 276 FEBS Journal 272 (2005) 269–281 ª 2004 FEBS [39] (data not shown), especially Arg52, which we con- sider to determine the substrate specificity. Structural basis for substrate stereorecognition and hydride transfer The structural analysis of HGDH and its comparison with other structurally characterized members of the D -2-hydroxyacid dehydrogenase family allows us to gain insight into the substrate specificity and reaction mechanism of HGDH. As mentioned earlier, there is broad substrate specificity among the members of the D -2-hydroxyacid dehydrogenase family. Therefore, cor- relations between substrate and active site properties can be inferred from their structural analysis. As expected by its substrate specificity towards lactate, the active site of LDH [15] has reduced dimensions with polar and hydrophobic character (Fig. 6A). As seen by the superposition of its active site with that from HGDH (Fig. 6B), the architecture of the two pockets is very dissimilar, correlating well with their unrelated substrates (pyruvate for LDH and 2-oxoglutarate for HGDH). While the LDH active site is small and dis- plays a general noncharged surface, the active site from HGDH is wider (even allowing that LDH is in the close conformation) and displays a generally posi- tively charged surface (Fig. 5B), optimized to bind the negatively charged 2-oxoglutarate molecule. A similar observation can be made from the comparison between the active sites from HicDH [16] (wide and with a gen- erally apolar surface, ideal to bind the 2-oxoisocapro- ate substrate; Fig. 6C) and HGDH (Figs 5B and 6D). One crucial event for catalysis in NADH-dependent dehydrogenases is the activation of the coenzyme, namely the enhancement of the electrophilicity of the nicotinamide moiety (at the pyridinium C4N atom containing the Re- and Si-hydrogen atoms) [40]. The coenzyme molecule is bound at the bottom wall of the interdomain cleft, sandwiched between the C-terminal ends of the b-strands bA, bB, bD–bF and helices aA and aC with the adenine moiety docked in a structur- ally conserved hydrophobic pocket (Val152, Ala206, Pro207, Val177, Ile209 and Val215 in HGDH). The adenine moiety interacts with a polar side chain (Asn212 in HGDH) and the pyrophosphate with a sol- vent molecule (HOH 1048 in HGDH), both structur- ally conserved. Additional interactions are possible with the consensus nucleotide-binding sequence motif [GXGX 2 GX 17 D(153–176) for HGDH], responsible for coenzyme discrimination [34]. Structural studies [11] have suggested that the coenzyme activation results from docking the B-side of the nicotinamide moiety against the hydrophobic patch built by aliphatic side chains (Pro103, Ile106, Ala107 and Ile157 in HGDH) and the backbone of the loop containing His297 (Leu298–Ser300 in HGDH). Additionally, hydrogen- bonding interactions between the carboxamide group and both a conserved aspartate (Asp259 in HGDH) and a backbone carbonyl group (Cys233 in HGDH) lock this group in the cis-conformation (related to the pyridinium ring). Both residues also play a role in the polarization of the C4N atom for hydride transfer [40,41]. Tyr101 can also participate as observed in FDH (its structural position is occupied by Asn146 [11]), LDH (Tyr202 [15]) and HicDH (Tyr100 [16]). A second important factor is the asymmetric induc- tion of the substrate [37], namely the correct orienta- tion of the substrate (Re- ⁄ Si-face) related to the coenzyme and proton-relay system, to originate a pro- ductive ternary complex. In HGDH the architecture of the active site with the arginine cluster (c-carboxylate group binding), the opposite polar patches and Arg235 (a-carboxylate group binding), and the proton-relay system Glu264-His297 is structurally optimized to recognize and fix the substrate for the correct stereo- chemical hydride transfer reaction (substrate’s Si-face opposite to the A-side of the coenzyme; Fig. 5). This structural optimization for substrate recognition and turnover is also displayed by other members of the protein family, e.g. LDH (Fig. 6A) and HicDH (Fig. 6C). The reduction of 2-oxoglutarate by HGDH is inhibited by the substrate analogue oxalylglycine (a weak competitive inhibitor; K i ¼ 5m M ). Substitution of the 3-methylene group by an amide (NH) can result in the formation of an extra hydrogen-bonding inter- action to the enzyme (probably Ser300), to yield an unproductive complex. L -Glutamate dehydrogenase [an NAD(P) + -dependent enzyme; EC 1.4.1.3] is also inhib- ited by oxalylglycine (K i ¼ 3–5 l M [42]). This different affinity results from the dissimilar active site environ- ments displayed by both enzymes (HGDH this study and L -glutamate dehydrogenase [43]). Interdomain cleft closure and catalysis The flexible character of the interdomain cleft permits the exchange between binding of both substrate and cofactor, their catalytic turnover and the release of product and cofactor ([19] and references within). In contrast to L -2-hydroxyacid dehydrogenases where the active site is shielded from solvent through the closure of the active site loop [44], in D -2-hydroxyacid dehy- drogenases (that lack such a loop) the solvent exclu- sion results from the closure of the interdomain cleft via rotation of the substrate-binding domain towards the nucleotide-binding domain as indicated by the B. M. Martins et al. NAD + -dependent (R)-2-hydroxyglutarate dehydrogenase FEBS Journal 272 (2005) 269–281 ª 2004 FEBS 277 structural analysis of apo and holoFDH [11]. However (and correlating with a common evolutionary origin for the two protein families) kinetic and structural studies [13] have indicated a sequential binding of coenzyme and substrate to the enzyme in its open con- formation followed by the closure of the interdomain cleft. In FDH, the structural ordering of the C-ter- minal helix and the rotation of helix aA observed upon coenzyme binding results in the interdomain cleft closure [11]. Helix aA is the major component of the dimer interface (helix aA in HGDH; Fig. 2A), explain- ing the structural rigidity observed for the nucleotide- binding domain when compared to the flexibility dis- played by the substrate-binding domain in the open and closed enzyme conformations (Fig. 3). When applied to HGDH, substrate may bind first to the wall of the cleft composed of the substrate-binding domain, where its c-carboxylate group would comple- ment the arginine cluster. Closure of the interdomain cleft would bring the substrate’s a-carboxylate group into the second anion binding site (Thr77–Gly79, Asp81, Tyr101, Ser300 and Arg235), resulting in sub- strate positioning between the coenzyme and the pro- ton-relay system His297-Glu264 and consequent solvent exclusion from the active site (Fig. 5). Alternat- ively, the interdomain cleft closure can result from the substrate movement towards the second anion-binding site. Whatever the driving force for the closure mech- anism is, the cooperative binding of substrate and coenzyme most certainly plays a role, as suggested by the absence of structures with substrate ⁄ inhibitor bound alone. The product (R)-2-hydroxyglutarate and oxidized coenzyme NAD + can exit by a reopening of the interdomain cleft. In addition to explaining the functionality of the catalytic triad, the structure–function relationships obtained by the comparison of HGDH with other struc- turally characterized members of the D -2-hydroxyacid dehydrogenase protein family give a chemically satisfy- ing view o f t he molecular mechanisms of substrate stereo- selectivity and of the hydride transfer reaction. Experimental procedures Protein purification and crystallization The cloning, sequencing and overexpression of the hgd gene from A. fermentans will be described elsewhere (J Bresser & W Buckel, unpublished results). Recombinant HGDH was purified to homogeneity by using previously described tech- niques [4] with minor modifications [2]. The purification protocol consisted of an ammonium sulfate precipitation (80%) followed by four chromatography steps (anionic exchange with DEAE, hydrophobic interaction with Phenyl- Sepharose, gel permeation with Superose-12 and a final polishing step with Mono-Q). Pure homogeneous HGDH (with a specific activity of 4800 UÆmg )1 [3]) was stored at 4 °C in a saturated solution of ammonium sulfate. HGDH was crystallised by the sitting drop vapour diffusion method at 4 °C. Prior to setting up the crystallization drops the protein was concentrated to 5 mgÆmL )1 and the buffer was exchanged to 5 m M Mops pH 7.0. The droplets were composed of 3 lL protein, 1 lL precipitant solution [15–18% (w ⁄ v) PEG MME 2000 or PEG 2000, and 0.2 M sodium or lithium acetate, pH 7.0–8.0] and 0.3–1.0 lL cofactor (NAD + ), substrate (2-oxoglutarate) or inhibitor (oxalylglycine, a substrate analogue) with final concentra- tions (per drop) of 5–10 m M . Polycrystals with a flower-like shape grew within 10–15 days to approximate dimensions of 0.7 · 0.4 · 0.4 mm. The crystals were split with the help of a thin needle or scalpel and were cryo-cooled in a nitrogen stream (Oxford Cryosystems, Oxford, UK) after immersion in a cryoprotectant solution [25–30% (w ⁄ v) PEG, 0.3 M salt (sodium or lithium acetate) and 10–15% (v ⁄ v) glycerol for 5–10 min. Heavy metal derivatization was con- ducted by soaking native crystals in a cryoprotectant solu- tion containing 2–10 m M of the heavy metals for 10–15 h. Data collection and processing Diffraction data were collected at the synchrotron beamline BW6 (DESY, Hamburg, Germany) at a crystal temperature of 100 K. The crystals diffracted to 1.98 A ˚ and belong to the hexagonal crystal system with cell dimensions of a ¼ b ¼ 67.49 A ˚ and c ¼ 312.78 A ˚ . Data were processed and scaled with DENZO and SCALEPACK [45]. The Matthews coef- ficient of 2.58 A ˚ 3 ÆDa )1 indicated a solvent content of 52% and one molecule per asymmetric unit. MAD data at the Ta L-III edge were collected from a Ta 6 Br 12 crystal deriv- ative to a resolution of 2.79 A ˚ (space group P622 or P6 x 22 with cell dimensions of a ¼ b ¼ 66.98 A ˚ and c ¼ 315.25 A ˚ ) and processed and scaled with XDS [46]. Structure determination and model refinement Patterson search techniques were performed with a search model comprising a truncated poly(Ala) chain with secon- dary structural elements conserved among the available structures of the D -2-hydroxyacid dehydrogenase protein family members. The search model accounted to approxi- mately 50% of the scattering power. The program AMORE [29] was used with data from 15 to 4 A ˚ and all six possible space groups (P622 and P6 1)5 22) were tested. For phase combination, first, the replacement model was subjected to a rigid body refinement against the MAD data using CNS [47] and model phases were calculated with REFMAC 5 [48]. Second, an anomalous Fourier map using the difference NAD + -dependent (R)-2-hydroxyglutarate dehydrogenase B. M. 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