Hydroxypyruvate Reductase

J. Mol. Biol. (2006) 360, 178–189

doi:10.1016/j.jmb.2006.05.018

Structural Basis of Substrate Specificity in Human Glyoxylate Reductase/Hydroxypyruvate Reductase Michael P. S. Booth 1 , R. Conners 1 , Gill Rumsby 2 and R. Leo Brady 1 ⁎ 1

Department of Biochemistry, University of Bristol, Bristol BS8 1TD, UK 2

Department of Clinical Biochemistry, UCL Hospitals, 60 Whitfield St., London, UK

Human glyoxylate reductase/hydroxypyruvate reductase (GRHPR) is a D2-hydroxy-acid dehydrogenase that plays a critical role in the removal of the metabolic by-product glyoxylate from within the liver. Deficiency of this enzyme is the underlying cause of primary hyperoxaluria type 2 (PH2) and leads to increased urinary oxalate levels, formation of kidney stones and renal failure. Here we describe the crystal structure of human GRHPR at 2.2 Å resolution. There are four copies of GRHPR in the crystallographic asymmetric unit: in each homodimer, one subunit forms a ternary (enzyme + NADPH + reduced substrate) complex, and the other a binary (enzyme + NADPH) form. The spatial arrangement of the two enzyme domains is the same in binary and ternary forms. This first crystal structure of a true ternary complex of an enzyme from this family demonstrates the relationship of substrate and catalytic residues within the active site, confirming earlier proposals of the mode of substrate binding, stereospecificity and likely catalytic mechanism for these enzymes. GRHPR has an unusual substrate specificity, preferring glyoxylate and hydroxypyruvate, but not pyruvate. A tryptophan residue (Trp141) from the neighbouring subunit of the dimer is projected into the active site region and appears to contribute to the selectivity for hydroxypyruvate. This first crystal structure of a human GRHPR enzyme also explains the deleterious effects of naturally occurring missense mutations of this enzyme that lead to PH2. © 2006 Elsevier Ltd All rights reserved.

*Corresponding author

Keywords: glyoxylate reductase; hydroxypyruvate reductase; hyperoxaluria; D-2-hydroxy-acid dehydrogenase; protein crystallography

Introduction The primary hyperoxalurias (PH) are genetic disorders of endogenous oxalate overproduction. Oxalate, particularly in the form of its calcium salt, is highly insoluble and the clinical consequences of excessive oxalate excretion (hyperoxaluria) are renal stone formation and/or nephrocalcinosis in child-

Abbreviations used: AGT, alanine:glyoxylate aminotransferase; D-GDH, D-glycerate dehydrogenase; D-HicDH, hydroxyisocaproate dehydrogenase; D-LDH, D-lactate dehydrogenase; GRHPR, glyoxylate reductase/hydroxypyruvate reductase; FDH, formate dehydrogenase; L-LDH, L-lactate dehydrogenase; PDB, Protein Data Bank (http://www.wwpdb.org); PH, primary hyperoxaluria; PH1, primary hyperoxaluria type 1; PH2, primary hyperoxaluria type 2; r.m.s.d., root mean square deviation. E-mail address of the corresponding author: [email protected]

hood leading to progressive renal damage, renal failure and reduced life expectancy. There are two well characterised forms of PH: PH1 and PH2, caused by deficiency of the enzymes alanine: glyoxylate aminotransferase (AGT) and glyoxylate reductase/hydroxypyruvate reductase (GRHPR), respectively (reviewed by Danpure 1 ). A third group, classified as ‘‘atypical’’ hyperoxaluria, has also been described2,3 but the underlying cause of this disorder(s) has yet to be defined. In both PH1 and PH2 the body’s ability to process the metabolic product glyoxylate is impaired. Glyoxylate is a highly reactive two carbon acid that is normally removed through its conversion to the amino acid glycine in the peroxisomes, catalysed by AGT, or by reduction to glycolate in the cytosol in a reaction catalysed by GRHPR (Figure 1). In the absence of either enzyme it appears that the competing oxidation of glyoxylate to oxalate by L-lactate dehydrogenase (L-LDH) dominates, leading to pronounced hyperoxaluria. It is thought that under normal conditions the

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Crystal Structure of GRHPR

179

Figure 1. Activity of human GRHPR and human A L-LDH on the related substrates glyoxylate, pyruvate and hydroxypyruvate. KM values for the substrates are all as reported4 and are all measured at pH 7.5.

competition between the reduction and oxidation of glyoxylate is finely balanced but controlled by the cytoplasmic levels of NADPH, which can be utilised by GRHPR but not L-LDH.1,4 Mutations in either the AGT or GRHPR genes disrupt this equilibrium, resulting in a build up of glyoxylate levels hence favouring its conversion to oxalate. Human GRHPR, found predominantly in the liver,5 also reduces the substrate hydroxypyruvate to D-glycerate (Figure 1). GRHPR is a homodimeric enzyme comprising 328 amino acid residues per subunit. On the basis of sequence homology GRHPR has been assigned to the D-2-hydroxy-acid dehydrogenase superfamily together with members for which structural information is available including D-lactate dehydrogenase (D-LDH), 6,7 D-glycerate dehydrogenase (D-GDH),8 phosphoglycerate dehydrogenase,9,10 hydroxyisocaproate dehydrogenase

(D-HicDH),11,12 NAD-dependent formate dehydrogenase (FDH),13,14 L-alanine dehydrogenase15 and the transcriptional co-repressor CtBP.16 Each subunit of a D-2-hydroxy-acid dehydrogenase comprises two distinctive α/β/α globular domains. These are referred to as the coenzyme-binding and the substrate-binding (or catalytic) domains, with the active site located in the cleft formed between the domains. Domain closure is believed to accompany binding of the co-enzyme, shielding the active site, in which a conserved histidine (FDH: His296) forms the acid/base catalyst and an invariant arginine (FDH: Arg235) orients the 2-hydroxy-acid substrates for catalysis, from solvent. The precise arrangement of substrates within the active sites throughout the family is inconsistently reported due to the use of a range of substrate analogues, and as many of the reported crystal structures comprise only apo or

180

Crystal Structure of GRHPR

holoenzyme forms. With reference to existing structures of D-2-hydroxy-acid dehydrogenases, GRHPR is expected to be most closely related to D-glycerate dehydrogenase from Hyphomicrobium methylovorum, which catalyses the same reaction with hydroxypyruvate. However, this bacterial enzyme has limited sequence homology (34%), differs in being exclusively NADH-dependent and its structure is only known in an apo (no co-enzyme, no substrate) form.8 Although the bacterial enzyme is referred to in the literature as D-glycerate dehydrogenase, in vivo both D-GDH and the human GRHPR enzyme function primarily as reductases leading to the formation of Dglycerate from hydroxypyruvate.8,17,18 The D-2-hydroxy-acid dehydrogenase fold differs from that observed for L-specific oxidoreductases archetypically represented by L-LDH, which competes with GRHPR for the same glyoxylate substrate (Figure 1). Both families of enzymes share a similar α/β/α fold with a central six-stranded sheet for the coenzyme-binding domains. In L-LDH, the second domain is closely associated with the co-enzymebinding domain and has an unusual and distinctive α + β fold,19 differing from the characteristic α/β/α topology found in the D-2-hydroxy-acid dehydrogenases. These substantial differences affect the spatial arrangement of substrate and co-enzyme when bound within the active site leading to differing stereo-specificities. GRHPR converts hydroxypyruvate to D-glycerate, whereas L-LDH interconverts the same substrate to L-glycerate. As the balance in the competition between GRHPR and L-LDH for glyoxylate is critical to our understanding of the biochemical consequences of PH2, the molecular basis of both substrate and co-enzyme selection by these enzymes is of interest. L-LDH is an archetypal oxido-reductase and has previously been extensively studied at the structural level (reviewed by Clarke & Dafform19). Here we describe for the first time the crystallographic structure of a human GRHPR enzyme, as an abortive ternary complex with both the reduced co-enzyme NADPH and reduced substrate D-glycerate.

Results Crystal structure of the GRHPR ternary complex Each monomer of GRHPR comprises two α/β/ α domains (Figure 2(a) and (b)), revealing the expected D-2-hydroxy-acid dehydrogenase fold predicted on the basis of sequence homology. Residues 107 to 298 form the larger, coenzyme-binding domain, comprising a core sheet of six strands flanked on one side by three and the other by four helices. This is a classical NAD(P) Rossmann fold, with the insertion of the extended dimer-forming loop between residues 123 and 149. The smaller domain, formed from residues 5–106 and 299–328, can be classified as a formate/glycerate dehydrogenase substrate-binding domain (SCOP 20 ). This

Figure 2. Overall structure of human GRHPR. (a) Cα trace of a single subunit, subunit A shown in blue; coenzyme and substrate in cyan and labelled. The amino and carboxyl termini are labelled N and C, respectively. (b) Cα trace of a dimer of GRHPR; subunit A is shown in blue, and subunit B in green. The Trp141 sidechain is shown in stick representation in yellow and is labelled; and the coenzyme and substrate are in cyan and also labelled. (c) Overlay of a single subunit of GRHPR (subunit A; blue) with CtBP16 (red), D-LDH6 (green), FDH14 (yellow) and bacterial D-GDH8 (cyan). Figures prepared with PyMol.41

Crystal Structure of GRHPR

181 Table 2. Comparison of human GRHPR monomers and dimers within asymmetric unit

domain has a flavodoxin-like fold with, once again, a central β-sheet but formed from five parallel strands with three helices on one side and two on the other. The coenzyme-binding domain in particular shows close structural homology with similar domains from other proteins in this family (Table 1) and, unsurprisingly, is most closely related to the bacterial DGDH structure.8 There is more variation in the Nterminal substrate binding domain, which matches most closely with the phosphoglycerate dehydrogenase structure,10 although this latter enzyme is an unusual member of this dehydrogenase family as it is tetrameric with three domains per monomer. The spatial relationship between the two domains is similar in all four copies of GRHPR in the crystallographic asymmetric unit (Table 2) with a small degree of bending at the hinge between the two domains noted for one of the subunits relative to the others. The inter-domain angles in the binary (holoenzyme) and ternary forms of GRHPR observed in this structure vary by about 18° from the arrangement observed for the apo form of the bacterial homologue (H. methylovorum D-GDH;8) and most closely match the ternary structure reported for CtBP16 and the apo form of formate dehydrogenase14 (Figure 2(c) and Table 1). Human GRHPR forms a homodimer in solution (data not shown). This is also seen in the crystal structure, where the asymmetric unit contains two dimers of GRHPR. The resulting staggered, ‘‘backto-back’’ oligomer in which all of the intermolecular contacts are made between residues from the coenzyme-binding domains is very similar to dimeric forms previously reported for other members of this family. In GRHPR this assembly is stabilised by a prominent extended helical (residues 123−136) and loop (residues 137−149) region that wraps around the neighbouring subunit. We refer to this segment as the ‘‘dimerisation loop’’ (Figure 2(c)). Significantly, the apex of this loop lies close to the active site of the other subunit (see below).

B C D AB

A

B

C

0.33 0.25 0.57

0.36 0.60

0.60

CD

0.62

Table shows the root mean square deviations in Å for the overlay of all main chain atoms from the subunits A−D and the dimers AB and CD.

Co-enzyme binding Although the NADPH predominantly makes contacts with the co-enzyme-binding domain, the binding site is located adjacent to the interface between the two domains (Figure 2(a)). NADPH coenzyme is seen to be bound to all four molecules of GRHPR in the crystallographic asymmetric unit, although with differing levels of occupancy. In the final model the co-enzyme has been included at 75% occupancy in two of the subunits; these are from separate biological dimers and correspond to the subunits in which the product molecule is also observed (see below). In the adjacent subunit of each dimer the electron density for the co-enzyme is of lower quality and the NADPH has been included in the final model at 50% occupancy for these sites. Neither of these subunits appear to have substrate or product bound. The active site Two of the molecules in the crystallographic asymmetric unit show the active site conformation of a ternary (enzyme + co-enzyme + substrate/product) complex (Figure 3(a) and (b)). The co-enzyme nicotinamide group is located and oriented as previously described for enzymes from this class. In addition, in both active sites there is clear density

Table 1. Comparison of GRHPR structure with other D-2-hydroxy-acid dehydrogenases Co-enzyme-binding domain

Structure 8

D-Glycerate dehydrogenase 6 D-Lactate dehydrogenase 6

D-Lactate dehydrogenase D-2-hydroxyisocaproate

dehydrogenase12 Transcription corepressor CtBP16 Formate dehydrogenase14 Formate dehydrogenase14 Phosphoglycerate dehydrogenase10

Substrate-binding domain

Overall monomer

Enzyme form

No. of equivalent Cα positions

r.m.s.d. (Å)

No. of equivalent Cα positions

r.m.s.d. (Å)

No. of equivalent Cα positions

r.m.s.d. (Å)

Apo Apo Holo + sulphate Ternary

187 172 178 171

1.3 1.6 1.5 1.4

125 124 123 121

2.0 2.2 2.3 2.5

294 273 290 278

2.4 2.7 2.1 2.9

Ternary

177

1.5

119

1.8

302

1.8

Apo Holo + sulphate Apo

184 184 178

1.6 1.6 1.5

121 123 123

2.1 2.1 1.5

260 261 283

1.9 2.1 2.8

Change in inter-domain angle a 18.5° 15.4°

8.9°

a Angle required to overlay substrate-binding domains of apo and binary/ternary structures after pre-alignment of the two coenzymebinding domains. For GRHPR/D-GDH, this value has been calculated by comparing the bacterial apo-D-GDH structure with the human ternary GRHPR structure.

Crystal Structure of GRHPR

182

Figure 3 (legend on opposite page)

Crystal Structure of GRHPR

for an oxalate-like molecule within the active site, although the presence of additional, contiguous density suggests that this is not oxalate that was preincubated with the enzyme prior to crystallisation. Placement of oxalate and a bound water molecule in the density did not lead to a model that could be successfully refined. Subsequent analysis by mass spectrometry (data not shown) of the oxalate sample used for these experiments indicated the oxalate contained a significant proportion (estimated ∼5%) of hydroxypyruvate, a known substrate for GRHPR. Both hydroxypyruvate and its reductive product Dglycerate match the observed electron density, and each could be readily incorporated within the refined model with similar and acceptable outcomes. However, as the enzyme was pre-incubated with the oxalate sample and reduced co-enzyme prior to crystallisation, all of the hydroxypyruvate is likely to have been converted to D -glycerate (NADPH was in molar excess). The combination of reduced co-enzyme with reduced product is also believed to be more likely to form an inhibitory complex. The electron density has therefore been assigned as two molecules of D-glycerate (reductive product) in the final model, and the ternary complex is believed to comprise a product inhibitory complex of enzyme/NADPH/D-glycerate. A comparison of the arrangement in the active site with structures described previously for enzymes from this family enables the critical residues for catalysis to be readily identified. By analogy with FDH,14 His293 (H296 in FDH) is expected to form the acid/base catalyst. The NE2 from the histidine sidechain is hydrogen bonded (2.9 Å) to the 2-hydroxy group of the D-glycerate substrate, the imidazole ring is both stabilised and orientated through a second hydrogen bond (2.8 Å) between its ND1 group and the Glu274 (FDH: E264) side-chain. This interaction is expected to raise the pKa of the imidazole ring. The product lies parallel to the nicotinamide group of the co-enzyme, sufficiently close to facilitate hydride transfer (3.0 Å from D-glycerate C2 to C4 of the nicotinamide). Arginine 269 (FDH: R235) holds and orientates the product through two charged hydrogen bonds (2.8 Å and 3.0 Å) made to the substrate 2hydroxyl and a single oxygen of the carboxylate group, respectively. The carboxylate oxygen atoms also form hydrogen bonds with the main chain nitrogen atoms of Val83 and Gly84. The product C3 atom lies in close proximity (3.3 Å) to and is believed to form van der Waals contacts with the CD2 methyl group of Leu59. The 3-hydroxyl group joined to this atom forms a hydrogen bond with the Ser296 sidechain and, via a conserved water molecule, to the imidazole nitrogen of the Trp141′ side-chain from

183 the adjacent subunit. This water molecule forms a network of interactions with the Arg302 guanadinium group, nicotinamide amide oxygen, and Ser296 side-chain, and is consistently observed to be bound in all four active sites. There are no significant changes in the arrangement of the amino acids within the active site between the binary and ternary complex forms of GRHPR (Figure 3(c)), with the substrate molecule replaced by solvent in the former. The product orientation and some of its interactions are similar to those previously reported in ternary complexes of D-hydroxyisocaproate dehydrogenase with the substrate analogue 2-oxo-4methylpentanoiuc acid11,12 and the CtBp1 protein with formate and acetate,16 but differ from that reported for L.heveticus D-LDH with oxamate (unpublished, but deposited in the Protein Data Bank (PDB), code: 2dld, 21 ) and the model proposed8 for the bacterial homologue D-GDHbinding hydroxypyruvate. Kinetic data Although pyruvate has previously been shown to be a poor substrate for bacterial D-GDH, there are no published references to the activity of human GRHPR towards this potential substrate. Kinetic analysis confirmed that human GRHPR also fails to substantially reduce this substrate. In the presence of NADPH, no activity was observed and the KM for pyruvate binding to GRHPR was found to exceed 100 mM.

Discussion Co-enzyme binding Differences in the angular variation between the two domains of D-2-hydroxy-acid dehydrogenases have been reported6,14 and have been noted to correspond with the binding of co-enzyme. As the co-enzyme binds at the confluence of the two domains, it is believed that hinge-bending motions (∼8° in FDH 14 , ∼16° in D -LDH 6 ) accompany binding, leading to enclosure of the substratebinding site. The crystal structure of human GRHPR reported here comprises two dimers of the enzyme. We observe a small angular difference between the subunits when one of the binary subunits is compared to the remaining subunits (Table 1), noting that all have co-enzyme bound to differing degrees. It seems likely that the varied co-

Figure 3. Arrangement of the active site of human GRHPR. (a) Stereoview showing D-glycerate, a fragment of NADPH, and active site residues (NADPH pink, D-glycerate cyan, residues from subunit A in green, residue from subunit B in yellow). (b) As for (a), but with the addition of 2Fobs−Fcalc electron density contoured at approximately 2 σ. (c) Stereoview of overlay of active site residues for binary and ternary complexes: subunit A + NADPH/D-glycerate (ternary complex) shown in blue; subunit B (binary complex) in red; subunit C (ternary complex) in green; subunit D (binary complex) in yellow. (d) Stereoview in similar orientation showing the conserved active site residues from human A LLDH42 in green, oxamate in cyan and NADH in pink.

184 enzyme occupancy between the subunits in the crystal most probably results from lattice packing restrictions. The overall temperature (B) factors are higher for one substrate-binding domain (chain D, Baverage = 43.8 Å2 for all main chain atoms; cf: 30.2 Å2 for the other three substrate-binding domains, and 30.0 Å2 for all co-enzyme-binding domains). This corresponds to the monomer in which the electron density for bound co-enzyme is of the poorest quality. This implies there is an inherent mobility of this domain, consistent with movement of the smaller catalytic domain on binary complex formation. We note that the ternary domain arrangement varies by an inter-domain rotation of about 18° from that reported for the bacterial homologue, D-GDH, which has been crystallised in its apo form. Human GRHPR may adopt a similar conformation in its apo form. However, considerable angular variability between the two domains is observed across this enzyme family (Figure 2(c)) and it is difficult to compare angular changes from one enzyme to another. Kinetic data show that GRHPR turnover of both hydroxypyruvate and glyoxylate proceeds with similar efficiency in the presence of either NADH or NADPH co-enzymes, although the latter is preferred (five to sixfold increase in specificity constant4). This distinguishes human GRHPR from its bacterial DGDH homologue. GRHPR binds the NADPH coNADPH 0.11 mM enzyme substantially more tightly (KM 4 NADH 2.42 mM ). Specificity for NADH over versus KM NADPH in dehydrogenases is usually achieved by the inclusion of an aspartic acid residue (Asp175 in L. bulgaricus D-LDH22) in the pocket normally occupied by the 2′-phosphate group in NADPH-dependent enzymes. This aspartic acid is not present in GRHPR, where the phosphate group is instead located in a pocket formed by the side-chains of arginine residues 185, 188 and glutamine 186. A salt-bridge and charged hydrogen bond formed with the two arginine residues appears to explain the approximately 20-fold tighter binding affinity of NADPH for this enzyme, while not excluding the binding of NADH at this site. Positively charged amino acids in this region have previously been noted to explain the preference of other oxido-reductases, such as the related short-chain dehydrogenases,23 for NADPH. Although specificity constants (kcat/KM) indicate that the efficiency of substrate turnover for GRHPR is not substantially diminished when NADH is used in place of NADPH, the ability of the enzyme to preferentially use this co-enzyme is likely to be critical in driving the reduction of glyoxylate in preference to its oxidation by LDH to oxalate. LDH is known to be almost exclusively dependent on NADH,19 implying that cellular NADPH concentrations determine the fate of glyoxylate in the presence of both GRHPR and LDH. Substrate binding The precise mode of substrate binding within the 2hydroxy-acid dehydrogenase family has remained a

Crystal Structure of GRHPR

source of some uncertainty, primarily due to the absence of crystal structures of true ternary complexes. Most of the existing crystal structures of complexes comprise either holoenzymes with additional anions such as sulphate bound within the active site (e.g. D-LDH + sulphate6, FDH + sulphate,14 CtBP1 + acetate16) or include substrate analogues of unclear relationship to the native substrates (DHicDH + ketoisocaproate, which competes with sulphate in the binding site24 and the CtBP1 complexes16). The structure of the bacterial GRHPR homologue D-GDH was determined in an apo form8 but used to model a ternary conformation based on the known ternary structure of L-LDH complexed with oxamate. In this model the C1−C2 bond of the substrate lies parallel with the C4−N axis of the nicotinamide ring (as seen for L-LDH in Figure 3(d)) being stabilised in this position by a salt-bridge between Arg240 and the two oxygen atoms of the substrate carboxylate. This model was supported in the deposited coordinates for the (unpublished) crystal structure of Lactobacillus helveticus D-LDH (PDB access code: 2DLD) in which an oxamate molecule is modelled in this conformation. This study was of limited resolution (2.7 Å). However, an alternative substrate-binding conformation for DLDH in which the arginine bridges a single oxygen from the carboxylate in addition to the reducible keto oxygen (as seen in Figure 3(a)) was later proposed,7 has been supported by mutational and kinetic data25 and is consistent with a model based on the binary crystal structure of Lactobacillus bulgaricus D-LDH[6]. In this arrangement the C3 methyl group of pyruvate is located within a distinctive hydrophobic pocket in the active site, and the si face of the substrate faces the nicotinamide ring, rather than the re face as in L-LDH (and hence accounting for the production of Dglycerate and L-glycerate, respectively). This orientation is observed in the crystal structure of the ternary complex of D-HicDH with NAD+ and its in vitro substrate ketoisocaproate,24 although interpretation of this structure is complicated by a sulphate ion that competes for substrate binding and the protein remains in an open (apo) conformation. Complexes of the transcription factor CtBP1 with coenzyme and acetate or formate bound in the active site have also been reported16 and are generally supportive of this second mode of binding, although these are very small ligands and their relationship to the unknown true substrate for this enzyme is unclear. Although this latter substrate orientation is now generally accepted, no direct crystallographic confirmation of this arrangement has been presented. The ternary structure reported here therefore provides the first direct evidence for the mode of substrate binding to GRHPR/D-GDH enzymes and, by analogy, to D-LDH and other D-2-hydroxy-acid dehydrogenases. The observed electron density for the substrate allowed us to unambiguously assign it as a C3substituted D-2-hydroxy-acid, with the bond lengths and interactions of the C3 substituent consistent with a hydroxyl group. The electron density was

Crystal Structure of GRHPR

therefore inconsistent with the expected structure of oxalate, which had been included in the crystallisation solution. The proximity of Leu59 to the C3 carbon also suggested that this density could not be attributed to a charged carboxylate oxygen. Identification of hydroxypyruvate in the sample therefore implied the presence in the crystal of either hydroxypyruvate or its reduced product, Dglycerate, both of which can be readily fitted to the electron density and refined. Although, in principle, it should be possible to distinguish these molecules from the C2 geometry, the resolution of the diffraction data prevents us from unambiguously making this assignment. However, as the reduced co-enzyme is present at approximately tenfold molar excess, it seems extremely unlikely that all of the hydroxypyruvate would not be turned over in the solution conditions and that therefore the complex trapped in the crystals most likely corresponds to the reduced co-enzyme and reduced substrate abortive complex of GRHPR/ NADPH/D-glycerate. Like many dehydrogenases, GRHPR has previously been noted to be inhibited in the presence of high concentrations of hydroxypyruvate substrate4, although further studies are required to clarify whether this is substrate or product inhibition. In both dimers of GRHPR only one of the two active sites is seen to contain the D-glycerate product molecule (refined, along with the NADPH, at 75% occupancy). It is unclear if this reflects an inherent property of the dimers themselves, or a restriction imposed by the crystal packing. Although not previously observed for the D-2-hydroxy-acid dehydrogenases, substrate occupancy of a single active site has previously been reported in the dimeric structure of malate dehydrogenase.26 Protrusion of the dimerisation loop into the active site pocket of the neighbouring subunit may provide a means to sense substrate occupancy, although it is not obvious from the structure how changes in the conformation of this region could be transmitted to the neighbouring monomer active site. An alternative explanation, suggested by the inherent thermal mobility noted for the substrate-binding domain of chain D, is that simultaneous closure of the two domains to form the active site in both subunits would be expected to have a higher entropic cost than that generated when only a single active site is occupied. The interactions observed between D-glycerate and the enzyme are consistent with those previously described in the D-HicDH/ketoisocaproate complex and as proposed for FDH and D-LDH enzymes. The 2-hydroxy-acid dehydrogenases characteristically contain a single conserved arginine within the active site, differing from L-LDH in which there are two. In the latter, Arg171 forms a bidentate salt-bridge with the substrate carboxylate group to bind and orient the substrate for catalysis (Figure 3(d)), whereas Arg109 from the active site loop is believed to polarize the bound substrate molecule to facilitate proton transfer.19 In D-LDH,

185 the single arginine (Arg245 in GRHPR) is placed to contribute both of these functions. It binds a single oxygen of the substrate carboxylate, thus contributing to orientation of the substrate, and additionally contacts the reducible keto oxygen, presumably leading to its polarisation. Additional substrate orientation is provided through both carboxylate oxygen atoms forming charged hydrogen bonds with the main chain amines of Val83 and Gly84. This arrangement has previously been observed in the D-HicDH/ketoisocaproate complex,24 and predicted for D-LDH.7 A recent mutational study28 of this loop region in Lactobacillus pentosus D-LDH demonstrated that the conformation of these residues was dependent on their interaction with an adjacent conserved asparagine (Asn97). Although it was predicted that the equivalent tyrosine residue in this location in GRHPR would similarly support the Val83–Gly84 loop, this proves not to be the case in the crystal structure of GRHPR where the tyrosine (Tyr102) side-chain points away from this loop. A single water molecule is also enclosed close to the active site in both the binary and ternary forms of GRHPR. This water makes multiple contacts to the protein: to the amine protons of the Trp141′ and Arg302 side-chains, to Ser296 and the carbonyl oxygen of the nicotinamide side-chain. Although as an aid to hydride transfer solvent is generally seen to be excluded from the active site of oxidoreductases on binding of substrate,19 enclosure of a single water molecule in a similar position has previously been noted in the crystal structure of Bacillus stearothermophilus L-LDH.27 This water too makes multiple contacts with the protein, perhaps ensuring its exclusion from the hydride transfer process. The inclusion of this solvent molecule within the active site of GRHPR may permit some flexibility to allow small adjustments for optimal binding of the differing substrates glyoxylate and hydroxypyruvate. Substrate specificity The selectivity of GRHPR is unusual. A comparison of the three potential substrates glyoxylate, pyruvate and hydroxypyruvate (Figure 1) shows that these differ by small (H-), medium (CH3-) and larger (HOCH2-) substituents, respectively, of the reducible carbonyl group. However, while both the small and larger substituents are tolerated by the enzyme, pyruvate forms a poor substrate for GRPHR (Figure 1) and for D-GDH.29 Assuming that the orientation of the D-glycerate product in the inhibitory complex observed in these crystals provides a good approximation for binding of these related molecules as substrates, the crystal structure provides an explanation for this unusual selectivity. The side-chain of Leu59 provides a steric restraint to the inclusion of substituents at the substrate C2 position. Hence, an absence of substituent (or the proton in glyoxylate) presents no barrier to binding and glyoxylate (KM = 0.24 mM4)

Crystal Structure of GRHPR

186 is a viable substrate. Inclusion of a bulky methyl group introduces a steric clash with the leucine sidechain; consequently pyruvate (KM > 100 mM) is a poor substrate. However, the larger hydroxymethyl group of hydroxypyruvate binds more tightly to the enzyme (KM = 0.058 mM4) than does glyoxylate. The location of D-glycerate in the enzyme active site indicates that the potential steric clash with the CD1 atom of Leu59 (which appears to form a van der Waals contact with the C3 of D-glycerate) can be compensated by the hydrogen bond to Ser296 and, via the conserved water molecule, to the imidazole nitrogen of Trp141′. This latter residue, intriguingly, is provided by the adjacent molecule in the dimer. The tryptophan is located on the periphery of the extended dimerisation loop, which is notably extended in both GRHPR and bacterial D-GDH relative to other 2-hydroxy-acid dehydrogenases (Figure 2(c)). This observation suggests that selectivity of GRHPR therefore appears to be dependent on the enzyme forming a dimer. This is consistent with the very strong conservation of both Ser296 and Trp141 in all known GRHPR sequences. Mutants of GRHPR Deficiency of GRHPR activity is known to underlie PH2, and a number of mutations5,30,31 have been identified in the gene for human GRHPR that lead to this condition. The crystal structure of GRHPR provides a basis for interpreting the effects of most of these mutations. As the NADP-binding domain is inserted within the sequence of the catalytic domain, the production of truncated forms of the protein is not viable and hence mutations introducing termination codons throughout the sequence will result in unfolded polypeptides. Four missense mutations with reduced catalytic activity have been reported, Gly160Arg, Gly165Asp, Arg302Cys and Met322Arg.5,31 The Gly160Arg mutation lies immediately adjacent to the adenine ribose ring. The bulk of the arginine side-chain at this position is likely to be detrimental for the binding of co-enzyme. Similarly, the Gly165Asp mutation is close to the centre of the co-enzyme-binding groove and lower affinity for the co-enzyme appears the likely cause of the reported reduction in catalytic activity (1.5% of wild-type5). The Met322Arg change most likely destabilises the structure of the C-terminal strand, which lies at the periphery of the channel leading to the active site. Two point mutations of Arg302 have been identified, to histidine (G.R., unpublished) and cysteine,5 this site being of particular interest for its proximity to the active site. This arginine is well conserved in GRHPR enzymes from various species. In both the binary and ternary complex forms the arginine sidechain forms a hydrogen bond to the conserved bound water, moderating its contacts with Ser296 and the imidazole amine of Trp141 from the adjacent subunit: the ‘‘gate-keeper’’ residue. Although both of the hydrophilic amino acids cysteine or histidine would appear to be readily incorporated within the

structure at this site, because of their smaller sizes they are unlikely to be capable of maintaining the network of interactions between the protein, conserved water and Ser296 and Trp141′ side-chains. The Arg302Cys mutation reduces but does not eliminate turnover of glyoxylate (5.6% activity) but abolishes hydroxypyruvate activity (<0.5% of wildtype 5 ). This observation is consistent with the proposal above that the intricate arrangement between the conserved bound water and Ser296 and Trp141′ side-chains is vital for regulating the binding of hydroxypyruvate substrate.

Conclusions On the basis of amino acid homology, GRHPR has previously been identified as a member of the D-2hydroxy-acid dehydrogenase family. This first crystal structure of human GRHPR confirms this prediction. GRHPR shows close structural similarity to D-GDH, FDH and other related dehydrogenases. This fortuitous ternary structure, formed by an inhibitory complex of reduced substrate with reduced co-enzyme, provides the first direct evidence of the active site arrangements of these enzymes with an actual substrate bound. The spatial arrangements of conserved residues within the active site explain the opposing stereo-specificities of GRHPR and L-LDH towards the substrate hydroxypyruvate. GRHPR also has an extended dimerisation loop that places a conserved tryptophan side-chain from one monomer in close proximity to the active site of the other monomer. Its placement is consistent with a role in discriminating between hydroxypyruvate, which is turned over efficiently by the enzyme, and pyruvate, which is not. This model therefore shows an unusual role for dimerisation in regulating substrate specificity for this enzyme. PH2 metabolic disease results from altered activity of human GRHPR, disrupting the equilibrium of the competition between GRHPR and LLDH for the substrate glyoxylate. The importance of this equilibrium is highlighted by the severe physiological consequences that arise when this balance is changed. As the crystal structures of human forms of both of these enzymes have now been determined, a detailed understanding of the molecular features that regulate this equilibrium is feasible.

Materials and Methods Expression, purification and crystallisation of GRHPR Human GRHPR was expressed in Escherichia coli (Rosetta strain, Invitrogen) cells using a modified form of an expression vector (pTrcHisB-HPR) described previously.4,32 The vector was modified to include an Nterminal fused thrombin cleavage sequence between the enzyme and hexa-histidine tag. After expression the cell pellet was resuspended in 20 mM Tris buffer (pH 8.5),

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500 mM NaCl, 20 mM imidazole, 10% (v/v) glycerol, 1 mM β-mercaptoethanol with EDTA-free protease inhibitor (Roche), loaded onto a HiTrap Ni2+ affinity column (Amersham Biosciences) and eluted with 0–1 M gradient of imidazole. The fusion protein was cleaved with thrombin (Amersham Biosciences) for 16 h at 18 °C followed by a benzamidine column (Amersham Biosciences) to remove the protease. The resulting cleaved GRHPR was buffer exchanged into 20 mM Tris (pH 8.5), 100 mM NaCl, 1 mM β-mercaptoethanol, concentrated (Vivaspin 10 kDa MWCO, Vivascience) and finally purified by gel filtration (HiLoad 16/60, Superdex 75 prep grade, Amersham Biosciences). Protein was >95% pure as judged by SDS-PAGE. Purified GRHPR was buffer exchanged into 20 mM Tris (pH 8.5) with 1 mM β-mercaptoethanol and concentrated (Centricon 10 kDa MWCO, Millipore) to 5.5 mg ml−1 as estimated by A280 nm absorbance. Concentrated protein was incubated on ice with 0.2 mM NADPH and 0.5 mM di-sodium oxalate (Sigma, Poole, UK), which was later found to contain a significant proportion of hydroxypyruvate, for 30 min prior to crystal tray setup. Crystals were grown by vapour-diffusion using 2 μl sitting-drops and mother liquor comprising 15% (w/v) PEG 8000, 0.2 M ammonium sulphate and 0.1 M sodium cacodylate (pH 6.5) at 18 °C. Crystals were not obtained with higher concentrations of NADPH or oxalate. Diffraction data collection X-ray diffraction data were collected at Daresbury SRS PX14.1 (λ = 1.448 Å) from crystals cooled to 100 K and cryoprotected in mother liquor with the addition of 30% glycerol. The data were processed and scaled in space group P21 using HKL2000.33 Poor crystal quality resulted in many reflections being discarded during processing, resulting in a relatively high Rmerge and limited completeness of the final data set (Table 3) including at low resolution (88% complete in the range 5−50 Å). The asymmetric unit contained four protein monomers and 53% (v/v) solvent as estimated using the Matthews Cell Content Analysis program in the CCP4 suite.34 Table 3. Summary of X-ray diffraction data and refined model statistics for human GRHPR crystal structure Space group Unit cell (Å) Resolution range (Å) Number of unique reflections I/σ Rmerge (%) Completeness (%) Redundancy Mosaicity (°) Refinement statistics Rcryst Rfree r.m.s.d. for bond length (Å) r.m.s.d. for bond angle (Å) Number of atoms in final model Quality of Ramachandran-plot Residues in most favoured regions (%) Residues in additional allowed regions (%) Residues in disallowed region (%)

P21 a = 76.6, b = 66.9, c = 149.8 β = 98.22 50−2.2 (2.28–2.2) 69,056 (5483) 8.4 (2.1) 12.1 (27.5) 90.3 (72.3) 2.6 (2.2) 0.586 0.201 0.282 0.017 1.866 10,701 88.4 11.1 0.4

Values in parentheses are the statistics for the highest resolution shell.

Structure solution and refinement The crystal structure of Hyphomicrobium methylovorum Dglycerate dehydrogenase (D-GDH, PDB code 1GDH)8 without solvent molecules was used as the molecular replacement model in Phaser version 1.3.35,36 The H. methylovorum D-GDH shares 34% sequence identity with human GRHPR. Searches using the whole enzyme, either as a dimer or monomer, failed. Further searches treated the two distinct domains – (the catalytic domain, residues 1 to 99 and 291 to 321, and the coenzyme binding domain, residues 100 to 290 (numbering as described for D-GDH8)) as separate search models. Searches using a dimeric model comprising the coenzyme-binding domains (100 to 290) produced two clear solutions (as expected) within the asymmetric unit. This solution was refined in Refmac537 initially using tight non-crystallographic symmetry restraints, mutated to the human sequence and rebuilt using COOT.38 A subsequent Phaser search incorporating models comprising the refined human coenzyme-binding domains and a poly-alanine model of the bacterial substrate-binding domain (1 to 99, 291 to 321) located good solutions for two of the expected four catalytic domains. After mutation of the substrate-binding domains to the human sequence and partial refinement, this model was used in a final Phaser search, which positively identified the orientation of the remaining two catalytic domains. Iterative cycles of model building in COOT and refinement with REFMAC5 were performed, with gradual removal of the non-crystallographic symmetry restraints as this was found to improve the R values. Four molecules of NADPH and two of D-glycerate were manually fitted into positive Fobs−Fcalc electron density. TLS Motion Determination39 was used to generate ten TLS groups for a final round of TLS refinement. The final model comprising 10,701 atoms including 610 water molecules and five sulphate molecules was monitored for geometrical correctness with PROCHECK40 and is summarised in Table 3. The model includes five non-glycine residues in the disallowed region of the Ramachandran plot: four of these are Asp105 from each chain, which has good electron density and appears to be distorted at the bend between the two domains. There is poor electron density for the remaining residue, Lys65 from chain D. Kinetic analyses The activity of human GRHPR towards the potential substrate pyruvate was measured using a spectrophotometer to monitor the conversion of NADPH to NADP+ using the procedure described.4 Assays were performed in 100 mM potassium phosphate buffer (pH 7.5). Protein Data Bank accession codes The coordinates and structure factors for crystal structure of GRHPR have been deposited in the RCSB Protein Data Bank with accession code 2GCG.

Acknowledgements M.P.S.B. is supported by an Overseas Research Studentship Award from Universities UK, and R.C.

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188 by grants from the Biotechnology and Biological Sciences Research Council UK. We are grateful to the staff at the Daresbury SRS synchrotron for access to facilities, and Dr Richard Sessions (University of Bristol) for helpful discussions.

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Edited by M. Guss (Received 14 March 2006; received in revised form 4 May 2006; accepted 8 May 2006) Available online 22 May 2006