Isolated eyelid neonatal Langerhans cell histiocytosis.

Langerhans cell histiocytosis (LCH) is a condition characterized by clonal proliferation of the phagocytic cells derived from the bone marrow. In this article, we present an exceedingly rare case of congenital/neonatal LCH in a 3-week-old girl who initially presented with an isolated swelling of the eyelid, initially misdiagnosed as a chalazion. Subsequently, a biopsy was performed, and histopathological evaluation confirmed the diagnosis of LCH. A staging work-up revealed no evidence of multisystem involvement, and thus, local steroid injection was performed as the initial treatment for the residual lesion. Cases of localized LCH that manifest as eyelid masses are rare, and most reported cases involve children over the age of one year. To the best of our knowledge, this case represents the first reported instance of neonatal LCH presenting as an eyelid mass. Although neonatal LCH is rare, ophthalmologists must be aware of this presentation and include it in the differential diagnosis for eyelid lesions in infants during the first month of life.

Discovery, Design, and Structural Characterization of Alkane-Producing Enzymes across the Ferritin-like Superfamily.

To complement established rational and evolutionary protein design approaches, significant efforts are being made to utilize computational modeling and the diversity of naturally occurring protein sequences. Here, we combine structural biology, genomic mining, and computational modeling to identify structural features critical to aldehyde deformylating oxygenases (ADOs), an enzyme family that has significant implications in synthetic biology and chemoenzymatic synthesis. Through these efforts, we discovered latent ADO-like function across the ferritin-like superfamily in various species of Bacteria and Archaea. We created a machine learning model that uses protein structural features to discriminate ADO-like activity. Computational enzyme design tools were then utilized to introduce ADO-like activity into the small subunit of Escherichia coli class I ribonucleotide reductase. The integrated approach of genomic mining, structural biology, molecular modeling, and machine learning has the potential to be utilized for rapid discovery and modulation of functions across enzyme families.

Structure and specificity of several triclocarban-binding single domain camelid antibody fragments.

The variable VHH domains of camelid single chain antibodies have been useful in numerous biotechnology applications due to their simplicity, biophysical properties, and abilities to bind to their cognate antigens with high affinities and specificity. Their interactions with proteins have been well-studied, but considerably less work has been done to characterize their ability to bind haptens. A high-resolution structural study of three nanobodies (T4, T9, and T10) which have been shown to bind triclocarban (TCC, 3-(4-chlorophenyl)-1-(3,4-dichlorophenyl)urea) with near-nanomolar affinity shows that binding occurs in a tunnel largely formed by CDR1 rather than a surface or lateral binding mode seen in other nanobody-hapten interactions. Additional significant interactions are formed with a non-hypervariable loop, sometimes dubbed "CDR4". A comparison of apo and holo forms of T9 and T10 shows that the binding site undergoes little conformational change upon binding of TCC. Structures of three nanobody-TCC complexes demonstrated there was not a standard binding mode. T4 and T9 have a high degree of sequence identity and bind the hapten in a nearly identical manner, while the more divergent T10 binds TCC in a slightly displaced orientation with the urea moiety rotated approximately 180° along the long axis of the molecule. In addition to methotrexate, this is the second report of haptens binding in a tunnel formed by CDR1, suggesting that compounds with similar hydrophobicity and shape could be recognized by nanobodies in analogous fashion. Structure-guided mutations failed to improve binding affinity for T4 and T9 underscoring the high degree of natural optimization.

Dopamine maintains network synchrony via direct modulation of gap junctions in the crustacean cardiac ganglion.

The Large Cell (LC) motor neurons of the crab cardiac ganglion have variable membrane conductance magnitudes even within the same individual, yet produce identical synchronized activity in the intact network. In a previous study we blocked a subset of K+ conductances across LCs, resulting in loss of synchronous activity (Lane et al., 2016). In this study, we hypothesized that this same variability of conductances makes LCs vulnerable to desynchronization during neuromodulation. We exposed the LCs to serotonin (5HT) and dopamine (DA) while recording simultaneously from multiple LCs. Both amines had distinct excitatory effects on LC output, but only 5HT caused desynchronized output. We further determined that DA rapidly increased gap junctional conductance. Co-application of both amines induced 5HT-like output, but waveforms remained synchronized. Furthermore, DA prevented desynchronization induced by the K+ channel blocker tetraethylammonium (TEA), suggesting that dopaminergic modulation of electrical coupling plays a protective role in maintaining network synchrony.

Conformational flexibility related to enzyme activity: evidence for a dynamic active-site gatekeeper function of Tyr(215) in Aerococcus viridans lactate oxidase.

L-Lactate oxidase (LOX) belongs to a large family of flavoenzymes that catalyze oxidation of α-hydroxy acids. How in these enzymes the protein structure controls reactivity presents an important but elusive problem. LOX contains a prominent tyrosine in the substrate binding pocket (Tyr(215) in Aerococcus viridans LOX) that is partially responsible for securing a flexible loop which sequesters the active site. To characterize the role of Tyr(215), effects of substitutions of the tyrosine (Y215F, Y215H) were analyzed kinetically, crystallographically and by molecular dynamics simulations. Enzyme variants showed slowed flavin reduction and oxidation by up to 33-fold. Pyruvate release was also decelerated and in Y215F, it was the slowest step overall. A 2.6-Å crystal structure of Y215F in complex with pyruvate shows the hydrogen bond between the phenolic hydroxyl and the keto oxygen in pyruvate is replaced with a potentially stronger hydrophobic interaction between the phenylalanine and the methyl group of pyruvate. Residues 200 through 215 or 216 appear to be disordered in two of the eight monomers in the asymmetric unit suggesting that they function as a lid controlling substrate entry and product exit from the active site. Substitutions of Tyr(215) can thus lead to a kinetic bottleneck in product release.

Speeding up the product release: a second-sphere contribution from Tyr191 to the reactivity of L-lactate oxidase revealed in crystallographic and kinetic studies of site-directed variants.

Among α-hydroxy acid-oxidizing flavoenzymes l-lactate oxidase (LOX) is unique in featuring a second-sphere tyrosine (Tyr191 in Aerococcus viridans LOX; avLOX) at the binding site for the substrate's carboxylate group. Y191F, Y191L and Y191A variants of avLOX were constructed to affect a hydrogen-bond network connecting Tyr191 to the carboxylate of the bound ligand via the conserved Tyr40 and to examine consequent effects on enzymatic reactivity. Kinetic studies at 20 °C and pH 6.5 revealed that release of pyruvate product was decreased 4.7-fold (Y191F), 19-fold (Y191L) and 28-fold (Y191A) compared with wild-type enzyme (~ 141 s(-1)) and thus became mainly rate limiting for l-lactate oxidation by the variants at a steady-state under air-saturated conditions. In the Y191L and the Y191A variants, but not in the Y191F variant, l-lactate binding was also affected strongly by the site-directed substitution. Reduction of the flavin cofactor by l-lactate and its reoxidation by molecular oxygen were, however, comparatively weakly affected by the replacements of Tyr191. Unlike the related lactate monooxygenase, which prevents the fast dissociation of pyruvate to promote its oxidative decarboxylation by H2 O2 into acetate, CO2 and water as final reaction products, all avLOX variants retained their native oxidase activity where catalytic turnover results in the equivalent formation of H2O2. The 1.9 Å crystal structure of the Y191F variant bound with FMN and pyruvate revealed a strictly locally disruptive effect of the site-directed substitution. Product off-rates appear to be dictated by partitioning of residues including Tyr191 from an active-site lid loop into bulk solvent and modulation of the hydrogen bond strength that links Tyr40 with the pyruvate's carboxylate group. Overall, this study emphasizes the possibly high importance of contributions from second-sphere substrate-binding residues to the fine-tuning of reactivity in α-hydroxy acid-oxidizing flavoenzymes, requiring that the catalytic steps of flavin reduction and oxidation are properly timed with the physical step of α-keto acid product release.

The Ala95-to-Gly substitution in Aerococcus viridans l-lactate oxidase revisited - structural consequences at the catalytic site and effect on reactivity with O2 and other electron acceptors.

Aerococcus viridansl-lactate oxidase (avLOX) is a biotechnologically important flavoenzyme that catalyzes the conversion of L-lactate and O2 into pyruvate and H2O2. The enzymatic reaction underlies different biosensor applications of avLOX for blood L-lactate determination. The ability of avLOX to replace O2 with other electron acceptors such as 2,6-dichlorophenol-indophenol (DCIP) allows the possiblity of analytical and practical applications. The A95G variant of avLOX was previously shown to exhibit lowered reactivity with O2 compared to wild-type enzyme and therefore was employed in a detailed investigation with respect to the specificity for different electron acceptor substrates. From stopped-flow experiments performed at 20 °C (pH 6.5), we determined that the A95G variant (fully reduced by L-lactate) was approximately three-fold more reactive towards DCIP (1.0 ± 0.1 × 10(6) M(-1) ·s(-1) ) than O2, whereas avLOX wild-type under the same conditions was 14-fold more reactive towards O2(1.8 ± 0.1 × 10(6) m(-1) ·s(-1)) than DCIP. Substituted 1,4-benzoquinones were up to five-fold better electron acceptors for reaction with L-lactate-reduced A95G variant than wild-type. A 1.65-Å crystal structure of oxidized A95G variant bound with pyruvate was determined and revealed that the steric volume created by removal of the methyl side chain of Ala95 and a slight additional shift in the main chain at position Gly95 together enable the accomodation of a new active-site water molecule within hydrogen-bond distance to the N5 of the FMN cofactor. The increased steric volume available in the active site allows the A95G variant to exhibit a similar trend with the related glycolate oxidase in electron acceptor substrate specificities, despite the latter containing an alanine at the analogous position.

Identification, characterization, and crystal structure of an aldo-keto reductase (AKR2E4) from the silkworm Bombyx mori.

A new member of the aldo-keto reductase (AKR) superfamily with 3-dehydroecdysone reductase activity was found in the silkworm Bombyx mori upon induction by the insecticide diazinon. The amino acid sequence showed that this enzyme belongs to the AKR2 family, and the protein was assigned the systematic name AKR2E4. In this study, recombinant AKR2E4 was expressed, purified to near homogeneity, and kinetically characterized. Additionally, its ternary structure in complex with NADP(+) and citrate was refined at 1.3Å resolution to elucidate substrate binding and catalysis. The enzyme is a 33-kDa monomer and reduces dicarbonyl compounds such as isatin and 17α-hydroxy progesterone using NADPH as a cosubstrate. No NADH-dependent activity was detected. Robust activity toward the substrate inhibitor 3-dehydroecdysone was observed, which suggests that this enzyme plays a role in regulation of the important molting hormone ecdysone. This structure constitutes the first insect AKR structure determined. Bound NADPH is located at the center of the TIM- or (ß/α)8-barrel, and residues involved in catalysis are conserved.

Crystallographic observation of pH-induced conformational changes in the Amyelois transitella pheromone-binding protein AtraPBP1.

The navel orangeworm, Amyelois transitella is a major agricultural pest causing large losses in a variety of tree crops. Control of this insect pest may be achieved by interfering with olfactory pathways to block detection of female-produced sex pheromones and consequently, disrupt mating. The first component of this pathway is the pheromone-binding protein AtraPBP1, which recognizes the pheromone and presents it to the odorant receptor housed in a sensory neuron of the male antennae. Release of the ligand depends on a pH-induced conformational change associated with the acidity of the membrane surface. To characterize this conformational change and to understand how pheromones bind, we have determined the high resolution crystal structures of AtraPBP1 in complex with two main constituents of the sex pheromone, i.e., (11Z,13Z)-hexadecadienal and (11Z,13Z)-hexadecadienol. Comparison with the structure of the unliganded form demonstrates a large ∼90° movement of the C-terminal helix which is observed in other pheromone- or odorant-binding proteins accompanied by an unpredicted 37° displacement of the N-terminal helix. Molecular dynamic trajectories suggest that the conformational change of the α1 helix facilitates the movement of the C-terminal helix.

Function of phenylalanine 259 and threonine 314 within the substrate binding pocket of the juvenile hormone esterase of Manduca sexta.

Juvenile hormone (JH) is a key insect developmental hormone that is found at low nanomolar levels in larval insects. The methyl ester of JH is hydrolyzed in many insects by an esterase that shows high specificity for JH. We have previously determined a crystal structure of the JH esterase (JHE) of the tobacco hornworm Manduca sexta (MsJHE) [Wogulis, M., Wheelock, C. E., Kamita, S. G., Hinton, A. C., Whetstone, P. A., Hammock, B. D., and Wilson, D. K. (2006) Biochemistry 45, 4045-4057]. Our molecular modeling indicates that JH fits very tightly within the substrate binding pocket of MsJHE. This tight fit places two noncatalytic amino acid residues, Phe-259 and Thr-314, within the appropriate distance and geometry to potentially interact with the alpha,beta-unsaturated ester and epoxide, respectively, of JH. These residues are highly conserved in numerous biologically active JHEs. Kinetic analyses of mutants of Phe-259 or Thr-314 indicate that these residues contribute to the low K(M) that MsJHE shows for JH. This low K(M), however, comes at the cost of reduced substrate turnover. Neither nucleophilic attack of the resonance-stabilized ester by the catalytic serine nor the availability of a water molecule for attack of the acyl-enzyme intermediate appears to be a rate-determining step in the hydrolysis of JH by MsJHE. We hypothesize that the release of the JH acid metabolite from the substrate binding pocket limits the catalytic cycle. Our findings also demonstrate that chemical bond strength does not necessarily correlate with how reactive the bond will be to metabolism.

Comprehensive X-ray structural studies of the quinolinate phosphoribosyl transferase (BNA6) from Saccharomyces cerevisiae.

Quinolinic acid phosphoribosyl transferase (QAPRTase, EC 2.4.2.19) is a 32 kDa enzyme encoded by the BNA6 gene in yeast and catalyzes the formation of nicotinate mononucleotide from quinolinate and 5-phosphoribosyl-1-pyrophosphate (PRPP). QAPRTase plays a key role in the tryptophan degradation pathway via kynurenine, leading to the de novo biosynthesis of NAD (+) and clearing the neurotoxin quinolinate. To improve our understanding of the specificity of the eukaryotic enzyme and the course of events associated with catalysis, we have determined the crystal structures of the apo and singly bound forms with the substrates quinolinate and PRPP. This reveals that the enzyme folds in a manner similar to that of various prokaryotic forms which are approximately 30% identical in sequence. In addition, the structure of the Michaelis complex is approximated by PRPP and the quinolinate analogue phthalate bound to the active site. These results allow insight into the kinetic mechanism of QAPRTase and provide an understanding of structural diversity in the active site of the Saccharomyces cerevisiae enzyme when compared to prokaryotic homologues.

Identification of formyl kynurenine formamidase and kynurenine aminotransferase from Saccharomyces cerevisiae using crystallographic, bioinformatic and biochemical evidence.

The essential enzymatic cofactor NAD+ can be synthesized in many eukaryotes, including Saccharomyces cerevisiae and mammals, using tryptophan as a starting material. Metabolites along the pathway or on branches have important biological functions. For example, kynurenic acid can act as an NMDA antagonist, thereby functioning as a neuroprotectant in a wide range of pathological states. N-Formyl kynurenine formamidase (FKF) catalyzes the second step of the NAD+ biosynthetic pathway by hydrolyzing N-formyl kynurenine to produce kynurenine and formate. The S. cerevisiae FKF had been reported to be a pyridoxal phosphate-dependent enzyme encoded by BNA3. We used combined crystallographic, bioinformatic and biochemical methods to demonstrate that Bna3p is not an FKF but rather is most likely the yeast kynurenine aminotransferase, which converts kynurenine to kynurenic acid. Additionally, we identify YDR428C, a yeast ORF coding for an alpha/beta hydrolase with no previously assigned function, as the FKF. We predicted its function based on our interpretation of prior structural genomics results and on its sequence homology to known FKFs. Biochemical, bioinformatics, genetic and in vivo metabolite data derived from LC-MS demonstrate that YDR428C, which we have designated BNA7, is the yeast FKF.

Structural and kinetic studies of induced fit in xylulose kinase from Escherichia coli.

The primary metabolic route for D-xylose, the second most abundant sugar in nature, is via the pentose phosphate pathway after a two-step or three-step conversion to xylulose-5-phosphate. Xylulose kinase (XK; EC 2.7.1.17) phosphorylates D-xylulose, the last step in this conversion. The apo and D-xylulose-bound crystal structures of Escherichia coli XK have been determined and show a dimer composed of two domains separated by an open cleft. XK dimerization was observed directly by a cryo-EM reconstruction at 36 A resolution. Kinetic studies reveal that XK has a weak substrate-independent MgATP-hydrolyzing activity, and phosphorylates several sugars and polyols with low catalytic efficiency. Binding of pentulose and MgATP to form the reactive ternary complex is strongly synergistic. Although the steady-state kinetic mechanism of XK is formally random, a path is preferred in which D-xylulose binds before MgATP. Modelling of MgATP binding to XK and the accompanying conformational change suggests that sugar binding is accompanied by a dramatic hinge-bending movement that enhances interactions with MgATP, explaining the observed synergism. A catalytic mechanism is proposed and supported by relevant site-directed mutants.

Catalytic mechanism and substrate selectivity of aldo-keto reductases: insights from structure-function studies of Candida tenuis xylose reductase.

Aldo-keto reductases (AKRs) constitute a large protein superfamily of mainly NAD(P)-dependent oxidoreductases involved in carbonyl metabolism. Catalysis is promoted by a conserved tetrad of active site residues (Tyr, Lys, Asp and His). Recent results of structure-function relationship studies for xylose reductase (AKR2B5) require an update of the proposed catalytic mechanism. Electrostatic stabilization by the epsilon-NH3+ group of Lys is a key source of catalytic power of xylose reductase. A molecular-level analysis of the substrate binding pocket of xylose reductase provides a case of how a very broadly specific AKR achieves the requisite selectivity for its physiological substrate and could serve as the basis for the design of novel reductases with improved specificities for biocatalytic applications.

On-bead combinatorial techniques for the identification of selective aldose reductase inhibitors.

Aldose reductase (AKR1B1; ALR2; E.C. 1.1.1.21) is an NADPH-dependent carbonyl reductase which has long been associated with complications resulting from the elevated blood glucose often found in diabetics. The development of effective inhibitors has been plagued by lack of specificity which has led to side effects in clinical trials. To address this problem, a library of bead-immobilized compounds was screened against fluorescently labeled aldose reductase in the presence of fluorescently labeled aldehyde reductase, a non-target enzyme, to identify compounds which were aldose reductase specific. Picked beads were decoded via novel bifunctional bead mass spec-based techniques and kinetic analysis of the ten inhibitors which were identified using this protocol yielded IC50 values in the micromolar range. Most importantly, all of these compounds showed a preference for aldose reductase with selectivities as high as approximately 7500-fold. The most potent of these exhibited uncompetitive inhibition versus the carbonyl-containing substrate D/L-glyceraldehyde with a Ki of 1.16 microM.

Identification of a novel NADH-specific aldo-keto reductase using sequence and structural homologies.

The AKRs (aldo-keto reductases) are a superfamily of enzymes which mainly rely on NADPH to reversibly reduce various carbonyl-containing compounds to the corresponding alcohols. A small number have been found with dual NADPH/NADH specificity, usually preferring NADPH, but none are exclusive for NADH. Crystal structures of the dual-specificity enzyme xylose reductase (AKR2B5) indicate that NAD+ is bound via a key interaction with a glutamate that is able to change conformations to accommodate the 2'-phosphate of NADP+. Sequence comparisons suggest that analogous glutamate or aspartate residues may function in other AKRs to allow NADH utilization. Based on this, nine putative enzymes with potential NADH specificity were identified and seven genes were successfully expressed and purified from Drosophila melanogaster, Escherichia coli, Schizosaccharomyces pombe, Sulfolobus solfataricus, Sinorhizobium meliloti and Thermotoga maritima. Each was assayed for co-substrate dependence with conventional AKR substrates. Three were exclusive for NADPH (AKR2E3, AKR3F2 and AKR3F3), two were dual-specific (AKR3C2 and AKR3F1) and one was specific for NADH (AKR11B2), the first such activity in an AKR. Fluorescence measurements of the seventh protein indicated that it bound both NADPH and NADH but had no activity. Mutation of the aspartate into an alanine residue or a more mobile glutamate in the NADH-specific E. coli protein converted it into an enzyme with dual specificity. These results show that the presence of this carboxylate is an indication of NADH dependence. This should allow improved prediction of co-substrate specificity and provide a basis for engineering enzymes with altered co-substrate utilization for this class of enzymes.

Structure-guided engineering of xylitol dehydrogenase cosubstrate specificity.

Xylitol dehydrogenase (XDH) is one of several enzymes responsible for assimilating xylose into eukaryotic metabolism and is useful for fermentation of xylose contained in agricultural byproducts to produce ethanol. For efficient xylose utilization at high flux rates, cosubstrates should be recycled between the NAD+-specific XDH and the NADPH-preferring xylose reductase, another enzyme in the pathway. To understand and alter the cosubstrate specificity of XDH, we determined the crystal structure of the Gluconobacter oxydans holoenzyme to 1.9 angstroms resolution. The structure reveals that NAD+ specificity is largely conferred by Asp38, which interacts with the hydroxyls of the adenosine ribose. Met39 stacked under the purine ring and was also located near the 2' hydroxyl. Based on the location of these residues and on sequence alignments with related enzymes of various cosubstrate specificities, we constructed a double mutant (D38S/M39R) that was able to exclusively use NADP+, with no loss of activity.

Structural studies of a potent insect maturation inhibitor bound to the juvenile hormone esterase of Manduca sexta.

Juvenile hormone (JH) is an insect hormone containing an alpha,beta-unsaturated ester consisting of a small alcohol and long, hydrophobic acid. JH degradation is required for proper insect development. One pathway of this degradation is through juvenile hormone esterase (JHE), which cleaves the JH ester bond to produce methanol and JH acid. JHE is a member of the functionally divergent alpha/beta-hydrolase family of enzymes and is a highly efficient enzyme that cleaves JH at very low in vivo concentrations. We present here a 2.7 A crystal structure of JHE from the tobacco hornworm Manduca sexta (MsJHE) in complex with the transition state analogue inhibitor 3-octylthio-1,1,1-trifluoropropan-2-one (OTFP) covalently bound to the active site. This crystal structure, the first JHE structure reported, contains a long, hydrophobic binding pocket with the solvent-inaccessible catalytic triad located at the end. The structure explains many of the interactions observed between JHE and its substrates and inhibitors, such as the preference for small alcohol groups and long hydrophobic backbones. The most potent JHE inhibitors identified to date contain a trifluoromethyl ketone (TFK) moiety and have a sulfur atom beta to the ketone. In this study, sulfur-aromatic interactions were observed between the sulfur atom of OTFP and a conserved aromatic residue in the crystal structure. Mutational analysis supported the hypothesis that these interactions contribute to the potency of sulfur-containing TFK inhibitors. Together, these results clarify the binding mechanism of JHE inhibitors and provide useful observations for the development of additional enzyme inhibitors for a variety of enzymes.

The crystal structure of an odorant binding protein from Anopheles gambiae: evidence for a common ligand release mechanism.

The Anopheles gambiae mosquito is the main vector of malaria transmission in sub-Saharan Africa. We present here a 1.5A crystal structure of AgamOBP1, an odorant binding protein (OBP) from the A. gambiae mosquito. The protein crystallized as a dimer with a unique binding pocket consisting of a continuous tunnel running through both subunits of the dimer and occupied by a PEG molecule. We demonstrate that AgamOBP1 undergoes a pH dependent conformational change that is associated with reduced ligand binding. A predominance of acid-labile hydrogen bonds involving the C-terminal loop suggests a mechanism in which a drop in pH causes C-terminal loop to open, leaving the binding tunnel solvent exposed, thereby lowering binding affinity for ligand. Because proteins from two distantly related insects also undergo a pH dependent conformational change involving the C-terminus that is associated with reduced ligand affinity, our results suggest a common mechanism for OBP activity.

Probing the substrate binding site of Candida tenuis xylose reductase (AKR2B5) with site-directed mutagenesis.

Little is known about how substrates bind to CtXR (Candida tenuis xylose reductase; AKR2B5) and other members of the AKR (aldo-keto reductase) protein superfamily. Modelling of xylose into the active site of CtXR suggested that Trp23, Asp50 and Asn309 are the main components of pentose-specific substrate-binding recognition. Kinetic consequences of site-directed substitutions of these residues are reported. The mutants W23F and W23Y catalysed NADH-dependent reduction of xylose with only 4 and 1% of the wild-type efficiency (kcat/K(m)) respectively, but improved the wild-type selectivity for utilization of ketones, relative to xylose, by factors of 156 and 471 respectively. Comparison of multiple sequence alignment with reported specificities of AKR members emphasizes a conserved role of Trp23 in determining aldehyde-versus-ketone substrate selectivity. D50A showed 31 and 18% of the wild-type catalytic-centre activities for xylose reduction and xylitol oxidation respectively, consistent with a decrease in the rates of the chemical steps caused by the mutation, but no change in the apparent substrate binding constants and the pattern of substrate specificities. The 30-fold preference of the wild-type for D-galactose compared with 2-deoxy-D-galactose was lost completely in N309A and N309D mutants. Comparison of the 2.4 A (1 A=0.1 nm) X-ray crystal structure of mutant N309D bound to NAD+ with the previous structure of the wild-type holoenzyme reveals no major structural perturbations. The results suggest that replacement of Asn309 with alanine or aspartic acid disrupts the function of the original side chain in donating a hydrogen atom for bonding with the substrate C-2(R) hydroxy group, thus causing a loss of transition-state stabilization energy of 8-9 kJ/mol.