Systematic strain construction and process development: Xylitol production by Saccharomyces cerevisiae expressing Candida tenuis xylose reductase in wild-type or mutant form.

A novel Saccharomyces cerevisiae whole-cell biocatalyst for xylitol production based on Candida tenuis xylose reductase (CtXR) is presented. Six recombinant strains expressing wild-type CtXR or an NADH-specific mutant were constructed and evaluated regarding effects of expression mode, promoter strength, biocatalyst concentration and medium composition. Intracellular XR activities ranged from 0.09 U mgProt(-1) to 1.05 U mgProt(-1) but did not correlate with the strains' xylitol productivities, indicating that other factors limited xylose conversion in the high-activity strains. The CtXR mutant decreased the biocatalyst's performance, suggesting use of the NADPH-preferring wild-type enzyme when (semi-)aerobic conditions are applied. In a bioreactor process, the best-performing strain converted 40 g L(-1) xylose with an initial productivity of 1.16 g L(-1)h(-1) and a xylitol yield of 100%. The obtained results underline the potential of CtXR wild-type for xylose reduction and point out parameters to improve "green" xylitol production.

Acceleration of an aldo-keto reductase by minimal loop engineering.

Aldo-keto reductases tighten coenzyme binding by forming a hydrogen bond across the pyrophosphate group of NAD(P)(H). Mutation of the hydrogen bonding anchor Lys24 in Candida tenuis xylose reductase prevents fastening of the "safety belt" around NAD(H). The loosened NAD(H) binding leads to increased turnover numbers (k(cat)) for reductions of bulky-bulky ketones at constant substrate and coenzyme affinities (i.e. K(m Ketone), K(m NADH)).

Stability and stabilization of D-amino acid oxidase from the yeast Trigonopsis variabilis.

The use of DAO (D-amino acid oxidase) for the conversion of cephalosporin C has provided a significant case for the successful implementation of an O(2)-dependent biocatalyst on an industrial scale. Improvement of the operational stability of the immobilized oxidase is, however, an important goal of ongoing process optimization. We have examined DAO from the yeast Trigonopsis variabilis with the aim of developing a rational basis for the stabilization of the enzyme activity at elevated temperature and under conditions of substrate turnover. Loss of activity in the resting enzyme can occur via different paths of denaturation. Partial thermal unfolding and release of the FAD cofactor, kinetically coupled with aggregation, contribute to the overall inactivation rate of the oxidase at 50 degrees C. Oxidation of Cys(108) into a stable cysteine sulfinic acid causes both decreased activity and stability of the enzyme. Strategies to counteract each of the denaturation steps in DAO are discussed. Fusion to a pull-down domain is a novel approach to produce DAO as protein-based insoluble particles that display high enzymatic activity per unit mass of catalyst.

Variations of the 2-His-1-carboxylate theme in mononuclear non-heme FeII oxygenases.

A facial triad of two histidine side chains and one aspartate or glutamate side chain forms the canonical metal-coordinating motif in the catalytic centers of various mononuclear non-heme Fe(II) enzymes. Although these active sites are based on totally unrelated protein folds and bring about a wide range of chemical transformations, most of them share the ability to couple dioxygen reduction with the oxygenation of an organic substrate. With the increasing number of protein structures now solved, it has become clear that the 2-His-1-carboxylate signature is less of a paradigm for non-heme Fe(II) active sites than had long been thought and that it can be replaced by alternative metal centers in various oxygenases, the structure-function relationships and proposed catalytic mechanisms of which are reviewed here. Metal coordination through three histidines and one glutamate constitutes the classical motif described for enzyme members of the cupin protein superfamily, such as aci-reductone dioxygenase and quercetin dioxygenase, multiple metal forms of which (including the Fe(II) type) are found in nature. Cysteine dioxygenase and diketone dioxygenase, which are strictly Fe(II)-dependent oxygenases based on the cupin fold, bind the catalytic metal through the homologous triad of histidines, but lack the fourth glutamate ligand. An alpha-ketoglutarate-dependent Fe(II) halogenase shows metal coordination by two histidines as the only protein-derived ligands, whilst carotene oxygenase, from a different protein fold family, features an Fe(II) site consisting of four histidine side chains. These recently discovered metallocenters are discussed with respect to their metal-binding properties and the reaction coordinates of the O(2)-dependent conversions they catalyze.

Alpha-retaining glucosyl transfer catalysed by trehalose phosphorylase from Schizophyllum commune: mechanistic evidence obtained from steady-state kinetic studies with substrate analogues and inhibitors.

Fungal trehalose phosphorylase is classified as a family 4 glucosyltransferase that catalyses the reversible phosphorolysis of alpha,alpha-trehalose with net retention of anomeric configuration. Glucosyl transfer to and from phosphate takes place by the partly rate-limiting interconversion of ternary enzyme-substrate complexes formed from binary enzyme-phosphate and enzyme-alpha-d-glucopyranosyl phosphate adducts respectively. To advance a model of the chemical mechanism of trehalose phosphorylase, we performed a steady-state kinetic study with the purified enzyme from the basidiomycete fungus Schizophyllum commune by using alternative substrates, inhibitors and combinations thereof in pairs as specific probes of substrate-binding recognition and transition-state structure. Orthovanadate is a competitive inhibitor against phosphate and alpha-d-glucopyranosyl phosphate, and binds 3 x 10(4)-fold tighter (K(i) approximately 1 microM) than phosphate. Structural alterations of d-glucose at C-2 and O-5 are tolerated by the enzyme at subsite +1. They lead to parallel effects of approximately the same magnitude (slope=1.14; r(2)=0.98) on the reciprocal catalytic efficiency for reverse glucosyl transfer [log (K(m)/k(cat))] and the apparent affinity of orthovanadate determined in the presence of the respective glucosyl acceptor (log K(i)). An adduct of orthovanadate and the nucleophile/leaving group bound at subsite +1 is therefore the true inhibitor and displays partial transition state analogy. Isofagomine binds to subsite -1 in the enzyme-phosphate complex with a dissociation constant of 56 microM and inhibits trehalose phosphorylase at least 20-fold better than 1-deoxynojirimycin. The specificity of the reversible azasugars inhibitors would be explained if a positive charge developed on C-1 rather than O-5 in the proposed glucosyl cation-like transition state of the reaction. The results are discussed in the context of alpha-retaining glucosyltransferase mechanisms that occur with and without a beta-glucosyl enzyme intermediate.

Galactosyl transfer catalyzed by thermostable beta-glycosidases from Sulfolobus solfataricus and Pyrococcus furiosus: kinetic studies of the reactions of galactosylated enzyme intermediates with a range of nucleophiles.

The transfer of a galactosyl group from an enzyme to a number of neutral primary alcohols, phenol and azide has been studied during the reactions at 80 degrees C of thermostable beta-glycosidases from Sulfolobus solfataricus (Ss beta Gly) and Pyrococcus furiosus (CelB) with 2-nitrophenyl beta-D-galactopyranoside or lactose (4-O-beta-D-galactopyranosyl D-glucopyranose) as substrates. The rate constant ratios, k(Nu)/k(water), for partitioning of the galactosylated enzyme intermediates between reaction with nucleophiles (k(Nu), M(-1) s(-1)) and water (k(water), s(-1)) have been determined from the difference in the initial velocities of the formation of 2-nitrophenol or D-glucose, and D-galactose. The results show that hydrophobic bonding interactions contribute approximately 8 kJ mol(-1) to the stabilization of the transition state for the reaction of galactosylated enzyme intermediates of Ss beta Gly and CelB with 1-butanol, compared to the transition state for the enzymatic reaction with methanol. The leaving group/nucleophile binding sites of Ss beta Gly and CelB appear about 0.8 times as hydrophobic as n-octanol. Values of k(Nu)/k(water) for reactions of galactosylated Ss beta Gly with ethanol and substituted derivatives of ethanol show no clear dependence on the pK(a) of the primary hydroxy group of these nucleophiles in the pK(a) range 12.4-16.0. The binding of phenol with the galactosylated enzyme intermediates of Ss beta Gly and CelB occurs in a form that is mainly nonproductive pertaining to beta-galactoside synthesis. Neither enzyme catalyzes galactosyl transfer to azide ion. A model is proposed for the interaction of neutral nucleophiles at an extended acceptor site of the galactosylated enzymes.

Transient-state and steady-state kinetic studies of the mechanism of NADH-dependent aldehyde reduction catalyzed by xylose reductase from the yeast Candida tenuis.

Microbial xylose reductase, a representative aldo-keto reductase of primary sugar metabolism, catalyzes the NAD(P)H-dependent reduction of D-xylose with a turnover number approximately 100 times that of human aldose reductase for the same reaction. To determine the mechanistic basis for that physiologically relevant difference and pinpoint features that are unique to the microbial enzyme among other aldo/keto reductases, we carried out stopped-flow studies with wild-type xylose reductase from the yeast Candida tenuis. Analysis of transient kinetic data for binding of NAD(+) and NADH, and reduction of D-xylose and oxidation of xylitol at pH 7.0 and 25 degrees C provided estimates of rate constants for the following mechanism: E + NADH right arrow over left arrow E.NADH right arrow over left arrow E.NADH + D-xylose right arrow over left arrow E.NADH.D-xylose right arrow over left arrow E.NAD(+).xylitol right arrow over left arrow E.NAD(+) right arrow over left arrow E.NAD(+) right arrow over left arrow E + NAD(+). The net rate constant of dissociation of NAD(+) is approximately 90% rate limiting for k(cat) of D-xylose reduction. It is controlled by the conformational change which precedes nucleotide release and whose rate constant of 40 s(-)(1) is 200 times that of completely rate-limiting E.NADP(+) --> E.NADP(+) step in aldehyde reduction catalyzed by human aldose reductase [Grimshaw, C. E., et al. (1995) Biochemistry 34, 14356-14365]. Hydride transfer from NADH occurs with a rate constant of approximately 170 s(-1). In reverse reaction, the E.NADH --> E.NADH step takes place with a rate constant of 15 s(-1), and the rate constant of ternary-complex interconversion (3.8 s(-1)) largely determines xylitol turnover (0.9 s(-1)). The bound-state equilibrium constant for C. tenuis xylose reductase is estimated to be approximately 45 (=170/3.8), thus greatly favoring aldehyde reduction. Formation of productive complexes, E.NAD(+) and E.NADH, leads to a 7- and 9-fold decrease of dissociation constants of initial binary complexes, respectively, demonstrating that 12-fold differential binding of NADH (K(i) = 16 microM) vs NAD(+) (K(i) = 195 microM) chiefly reflects difference in stabilities of E.NADH and E.NAD(+). Primary deuterium isotope effects on k(cat) and k(cat)/K(xylose) were, respectively, 1.55 +/- 0.09 and 2.09 +/- 0.31 in H(2)O, and 1.26 +/- 0.06 and 1.58 +/- 0.17 in D(2)O. No deuterium solvent isotope effect on k(cat)/K(xylose) was observed. When deuteration of coenzyme selectively slowed the hydride transfer step, (D)()2(O)(k(cat)/K(xylose)) was inverse (0.89 +/- 0.14). The isotope effect data suggest a chemical mechanism of carbonyl reduction by xylose reductase in which transfer of hydride ion is a partially rate-limiting step and precedes the proton-transfer step.

Exploring the active site of yeast xylose reductase by site-directed mutagenesis of sequence motifs characteristic of two dehydrogenase/reductase family types.

Starting from a common tyrosine, yeast xylose reductases (XRs) contain two conserved sequence motifs corresponding to the catalytic signatures of single-domain reductases/epimerases/dehydrogenases (Tyr(n)-(X)3-Lys(n+4)) and aldo/keto reductases (AKRs) (Tyr(n)-(X)28-Lys(n+29)). Tyr(51), Lys(55) and Lys(80) of XR from Candida tenuis were replaced by site-directed mutagenesis. The purified Tyr(51)--> Phe and Lys(80)-->Ala mutants showed turnover numbers and catalytic efficiencies for NADH-dependent reduction of D-xylose between 2500- and 5000-fold below wild-type levels, suggesting a catalytic role of both residues. Replacing Lys(55) by Asn, a substitution found in other AKRs, did not detectably affect binding of coenzymes, and enzymatic catalysis to carbonyl/alcohol interconversion. The contribution of Tyr(51) to rate enhancement of aldehyde reduction conforms with expectations for the general acid catalyst of the enzymatic reaction.

Fungal trehalose phosphorylase: kinetic mechanism, pH-dependence of the reaction and some structural properties of the enzyme from Schizophyllum commune.

Initial-velocity measurements for the phospholysis and synthesis of alpha,alpha-trehalose catalysed by trehalose phosphorylase from Schizophyllum commune and product and dead-end inhibitor studies show that this enzyme has an ordered Bi Bi kinetic mechanism, in which phosphate binds before alpha,alpha-trehalose, and alpha-D-glucose is released before alpha-D-glucose 1-phosphate. The free-energy profile for the enzymic reaction at physiological reactant concentrations displays its largest barriers for steps involved in reverse glucosyl transfer to D-glucose, and reveals the direction of phospholysis to be favoured thermodynamically. The pH dependence of kinetic parameters for all substrates and the dissociation constant of D-glucal, a competitive dead-end inhibitor against D-glucose (K(i)=0.3 mM at pH 6.6 and 30 degrees C), were determined. Maximum velocities and catalytic efficiencies for the forward and reverse reactions decrease at high and low pH, giving apparent pK values of 7.2--7.8 and 5.5--6.0 for two groups whose correct protonation state is required for catalysis. The pH dependences of k(cat)/K are interpreted in terms of monoanionic phosphate and alpha-D-glucose 1-phosphate being the substrates, and of the pK value seen at high pH corresponding to the phosphate group in solution or bound to the enzyme. The K(i) value for the inhibitor decreases outside the optimum pH range for catalysis, indicating that binding of D-glucal is tighter with incorrectly ionized forms of the complex between the enzyme and alpha-D-glucose 1-phosphate. Each molecule of trehalose phosphorylase contains one Mg(2+) that is non-dissociable in the presence of metal chelators. Measurements of the (26)Mg(2+)/(24)Mg(2+) ratio in the solvent and on the enzyme by using inductively coupled plasma MS show that exchange of metal ion between protein and solution does not occur at measurable rates. Tryptic peptide mass mapping reveals close structural similarity between trehalose phosphorylases from basidiomycete fungi.

Structural and functional properties of aldose xylose reductase from the D-xylose-metabolizing yeast Candida tenuis.

The primary structure of the aldose xylose reductase from Candida tenuis (CtAR) is shown to be 39% identical to that of human aldose reductase (hAR). The catalytic tetrad of hAR is completely conserved in CtAR (Tyr51, Lys80, Asp46, His113). The amino acid residues involved in binding of NADPH by hAR (D.K. Wilson, et al., Science 257 (1992) 81-84) are 64% identical in CtAR. Like hAR the yeast enzyme is specific for transferring the 4-pro-R hydrogen of the coenzyme. These properties suggest that CtAR is a member of the aldo/keto reductase superfamily. Unlike hAR the enzyme from C. tenuis has a dual coenzyme specificity and shows similar specificity constants for NADPH and NADH. It binds NADP(+) approximately 250 times less tightly than hAR. Typical turnover numbers for aldehyde reduction by CtAR (15-20 s(-1)) are up to 100-fold higher than corresponding values for hAR, probably reflecting an overall faster dissociation of NAD(P)(+) in the reaction catalyzed by the yeast enzyme.

Role of non-covalent enzyme-substrate interactions in the reaction catalysed by cellobiose phosphorylase from Cellulomonas uda.

Steady-state kinetic studies of the enzymic glucosyl transfer to and from phosphate catalysed by cellobiose phosphorylase from Cellulomonas uda have shown that this enzyme operates by a ternary-complex kinetic mechanism in which beta-cellobiose binds before phosphate, and beta-D-glucose and alpha-D-glucopyranosyl phosphate are released in that order. alpha-D-Glucopyranosyl fluoride (but not beta-D-glucopyranosyl fluoride) serves as alternative glucosyl donor for beta-cellobiose synthesis with a specificity constant that is one-ninth that of the corresponding enzymic reaction with alpha-D-glucopyranosyl phosphate (approximately 20000 M(-1).s(-1) at 30 degrees C). The kinetic parameters for a complete series of deoxy and deoxyfluoro analogues of D-glucose have been determined and the data yield estimates of the net strengths of hydrogen-bonding interactions with the non-reacting hydroxy groups of D-glucose at the transition state (0.8-4.0 kcal/mol, where 1 cal identical with 4.184 J) and enable the prediction of the polarities of these hydrogen bonds. Each hydroxy group functions as donor of a hydrogen for bonding to probably a charged (at 3-OH) or neutral (at 2-OH and 6-OH) acceptor group on the enzyme. The equatorial 1-OH is essential for enzyme activity. Derivatives of D-glucose in which the 1-OH or the reacting 4-OH were replaced by hydrogen or fluorine have been tested as inhibitors to measure their affinities for the sugar-binding subsite +1 (numbered from the bond-cleaving/forming site). The data show that hydrogen-bonding interactions between the 1-OH and 4-OH and charged groups on the enzyme stabilize the ground-state ternary complex of the enzymic synthesis of beta-cellobiose by 2.3 and 0.4 kcal/mol, respectively, and assist the precise positioning of beta-D-glucose for catalysis.

Transgalactosylation by thermostable beta-glycosidases from Pyrococcus furiosus and Sulfolobus solfataricus. Binding interactions of nucleophiles with the galactosylated enzyme intermediate make major contributions to the formation of new beta-glycosides during lactose conversion.

The hyperthermostable beta-glycosidases from the Archaea Sulfolobus solfataricus (SsbetaGly) and Pyrococcus furiosus (CelB) hydrolyse beta-glycosides of D-glucose or D-galactose with relaxed specificities pertaining to the nature of the leaving group and the glycosidic linkage. To determine how specificity is manifested under conditions of kinetically controlled transgalactosylation, the major transfer products formed during the hydrolysis of lactose by these enzymes have been identified, and their appearance and degradation have been determined in dependence of the degree of substrate conversion. CelB and SsbetaGly show a marked preference for making new beta(1-->3) and beta(1-->6) glycosidic bonds by intermolecular as well as intramolecular transfer reactions. The intramolecular galactosyl transfer of CelB, relative to glycosidic-bond cleavage and release of glucose, is about 2.2 times that of SsbetaGly and yields beta-D-Galp-(1-->6)-D-Glc and beta-D-Galp-(1-->3)-D-Glc in a molar ratio of approximately 1 : 2. The partitioning of galactosylated SsbetaGly between reaction with sugars [kNu (M-1. s-1)] and reaction with water [kwater (s-1)] is about twice that of CelB. It gives a mixture of linear beta-D-glycosides, chiefly trisaccharides at early reaction times, in which the prevailing new glycosidic bonds are beta(1-->6) and beta(1-->3) for the reactions catalysed by SsbetaGly and CelB, respectively. The accumulation of beta-D-Galp-(1-->6)-D-Glc at the end of lactose hydrolysis reflects a 3-10-fold specificity of both enzymes for the hydrolysis of beta(1-->3) over beta(1-->6) linked glucosides. Galactosyl transfer from SsbetaGly or CelB to D-glucose occurs with partitioning ratios, kNu/kwater, which are seven and > 170 times those for the reactions of the galactosylated enzymes with 1-propanol and 2-propanol, respectively. Therefore, the binding interactions with nucleophiles contribute chiefly to formation of new beta-glycosides during lactose conversion. Likewise, noncovalent interactions with the glucose leaving group govern the catalytic efficiencies for the hydrolysis of lactose by both enzymes. They are almost fully expressed in the rate-limiting first-order rate constant for the galactosyl transfer from the substrate to the enzyme and lead to a positive deviation by approximately 2.5 log10 units from structure-reactivity correlations based on the pKa of the leaving group.

Thermal denaturation pathway of starch phosphorylase from Corynebacterium callunae: oxyanion binding provides the glue that efficiently stabilizes the dimer structure of the protein.

Starch phosphorylase from Corynebacterium callunae is a dimeric protein in which each mol of 90 kDa subunit contains 1 mol pyridoxal 5'-phosphate as an active-site cofactor. To determine the mechanism by which phosphate or sulfate ions bring about a greater than 500-fold stabilization against irreversible inactivation at elevated temperatures (> or = 50 degrees C), enzyme/oxyanion interactions and their role during thermal denaturation of phosphorylase have been studied. By binding to a protein site distinguishable from the catalytic site with dissociation constants of Ksulfate = 4.5 mM and Kphosphate approximately 16 mM, dianionic oxyanions induce formation of a more compact structure of phosphorylase, manifested by (a) an increase by about 5% in the relative composition of the alpha-helical secondary structure, (b) reduced 1H/2H exchange, and (c) protection of a cofactor fluorescence against quenching by iodide. Irreversible loss of enzyme activity is triggered by the release into solution of pyridoxal 5'-phosphate, and results from subsequent intermolecular aggregation driven by hydrophobic interactions between phosphorylase subunits that display a temperature-dependent degree of melting of secondary structure. By specifically increasing the stability of the dimer structure of phosphorylase (probably due to tightened intersubunit contacts), phosphate, and sulfate, this indirectly (1) preserves a functional active site up to approximately 50 degrees C, and (2) stabilizes the covalent protein cofactor linkage up to approximately 70 degrees C. The effect on thermostability shows a sigmoidal and saturatable dependence on the concentration of phosphate, with an apparent binding constant at 50 degrees C of approximately 25 mM. The extra stability conferred by oxyanion-ligand binding to starch phosphorylase is expressed as a dramatic shift of the entire denaturation pathway to a approximately 20 degrees C higher value on the temperature scale.

Development of an ultra-high-temperature process for the enzymatic hydrolysis of lactose: II. Oligosaccharide formation by two thermostable beta-glycosidases.

During lactose conversion at 70 degrees C, when catalyzed by beta-glycosidases from the archea Sulfolobus solfataricus (SsbetaGly) and Pyrococcus furiosus (CelB), galactosyl transfer to acceptors other than water competes efficiently with complete hydrolysis of substrate. This process leads to transient formation of a range of new products, mainly disaccharides and trisaccharides, and shows a marked dependence on initial substrate concentration and lactose conversion. Oligosaccharides have been analyzed quantitatively by using capillary electrophoresis and high performance anion-exchange chromatography. At 270 g/L initial lactose, they accumulate at a maximum concentration of 86 g/L at 80% lactose conversion. With both enzymes, the molar ratio of trisaccharides to disaccharides is maximal at an early stage of reaction and decreases directly proportional to increasing substrate conversion. Overall, CelB produces about 6% more hydrolysis byproducts than SsbetaGly. However, the product spectrum of SsbetaGly is richer in trisaccharides, and this agrees with results obtained from the steady-state kinetics analyses of galactosyl transfer catalyzed by SsbetaGly and CelB. The major transgalactosylation products of SsbetaGly and CelB have been identified. They are beta-D-Galp-(1-->3)-Glc and beta-D-Galp-(1-->6)-Glc, and beta-D-Galp-(1-->3)-lactose and beta-D-Galp-(1-->6)-lactose, and their formation and degradation have been shown to be dependent upon lactose conversion. Both enzymes accumulate beta(1-->6)-linked glycosides, particularly allolactose, at a late stage of reaction. Because a high oligosaccharide concentration prevails until about 80% lactose conversion, thermostable beta-glycosidases are efficient for oligosaccharide production from lactose. Therefore, they prove to be stable and versatile catalysts for lactose utilization.

Mechanism of thermal denaturation of maltodextrin phosphorylase from Escherichia coli.

Maltodextrin phosphorylase from Escherichia coli (MalP) is a dimeric protein in which each approximately 90-kDa subunit contains active-site pyridoxal 5'-phosphate. To unravel factors contributing to the stability of MalP, thermal denaturations of wild-type MalP and a thermostable active-site mutant (Asn-133-->Ala) were compared by monitoring enzyme activity, cofactor dissociation, secondary structure content and aggregation. Small structural transitions of MalP are shown by Fourier-transform infrared spectroscopy to take place at approximately 45 degrees C. They are manifested by slight increases in unordered structure and (1)H/(2)H exchange, and reflect reversible inactivation of MalP. Aggregation of the MalP dimer is triggered by these conformational changes and starts at approximately 45 degrees C without prior release into solution of pyridoxal 5'-phosphate. It is driven by electrostatic rather than hydrophobic interactions between MalP dimers, and leads to irreversible inactivation of the enzyme. Aggregation is inhibited efficiently and specifically by oxyanions such as phosphate, and AMP which therefore, stabilize MalP against the irreversible denaturation step at 45 degrees C. Melting of the secondary structure in soluble and aggregated MalP takes place at much higher temperatures of approx. 58 and 67 degrees C, respectively. Replacement of Asn-133 by Ala does not change the mechanism of thermal denaturation, but leads to a shift of the entire pathway to a approximately 15 degrees C higher value on the temperature scale. Apart from greater stability, the Asn-133-->Ala mutant shows a 2-fold smaller turnover number and a 4.6-fold smaller energy of activation than wild-type MalP, probably indicating that the site-specific replacement of Asn-133 brings about a greater rigidity of the active-site environment of the enzyme. A structure-based model is proposed which explains the stabilizing interaction between MalP and oxyanions, or AMP.

D-Xylose metabolism by Candida intermedia: isolation and characterisation of two forms of aldose reductase with different coenzyme specificities.

To study individual enzyme components responsible for the initial step of D-xylose utilisation by the yeast Candida intermedia, a two-step protocol has been developed that enables clear-cut separation and isolation of two structurally similar but functionally different aldose reductases (ALRs) in high yield. In the first step, the yeast cell extract is fractionated efficiently by biomimetic chromatography using the dye HE-3B (reactive Red 120) as pseudoaffinity ligand coupled to Sepharose CL-4B. In the second step, optimised high-resolution anion-exchange chromatography using Mono Q yields purified ALR1 and ALR2 in overall yields of 63 and 62%, respectively. ALR1 is strictly specific for NADPH (2.4 x 10(5) M(-1) s(-1)) whereas ALR2 utilises NADH and NADPH with similar specificity constants of approximately 2-4 x 10(5) M(-1) s(-1). Both enzymes are dimers with a subunit molecular mass of 36000 but they differ in pI and the number of titratable sulphydryl groups in the native protein. The chromatographic procedure identifies microheterogeneity in recombinant aldose reductase from Candida tenuis overexpressed in Escherichia coli.

Binding energy and specificity in the catalytic mechanism of yeast aldose reductases.

Derivatives of d-xylose and d-glucose, in which the hydroxy groups at C-5, and C-5 and C-6 were replaced by fluorine, hydrogen and azide, were synthesized and used as substrates of the NAD(P)H-dependent aldehyde reduction catalysed by aldose reductases isolated from the yeasts Candida tenuis, C. intermedia and Cryptococcus flavus. Steady-state kinetic analysis showed that, in comparison with the parent aldoses, the derivatives were reduced with up to 3000-fold increased catalytic efficiencies (k(cat)/K(m)), reflecting apparent substrate binding constants (K(m)) decreased to as little as 1/250 and, for d-glucose derivatives, up to 5.5-fold increased maximum initial rates (k(cat)). The effects on K(m) mirror the relative proportion of free aldehyde that is available in aqueous solution for binding to the binary complex enzyme-NAD(P)H. The effects on k(cat) reflect non-productive binding of the pyranose ring of sugars; this occurs preferentially with the NADPH-dependent enzymes. No transition-state stabilization energy seems to be derived from hydrogen-bonding interactions between enzyme-NAD(P)H and positions C-5 and C-6 of the aldose. In contrast, unfavourable interactions with the C-6 group are used together with non-productive binding to bring about specificity (6-10 kJ/mol) in a series of d-aldoses and to prevent the reaction with poor substrates such as d-glucose. Azide introduced at C-5 or C-6 destabilizes the transition state of reduction of the corresponding hydrogen-substituted aldoses by approx. 4-9 kJ/mol. The total transition state stabilization energy derived from hydrogen bonds between hydroxy groups of the substrate and enzyme-NAD(P)H is similar for all yeast aldose reductases (yALRs), at approx. 12-17 kJ/mol. Three out of four yALRs manage on only hydrophobic enzyme-substrate interactions to achieve optimal k(cat), whereas the NAD(P)H-dependent enzyme from C. intermedia requires additional, probably hydrogen-bonding, interactions with the substrate for efficient turnover.

Kinetic study of the catalytic mechanism of mannitol dehydrogenase from Pseudomonas fluorescens.

To characterize catalysis by NAD-dependent long-chain mannitol 2-dehydrogenases (MDHs), the recombinant wild-type MDH from Pseudomonas fluorescens was overexpressed in Escherichia coli and purified. The enzyme is a functional monomer of 54 kDa, which does not contain Zn(2+) and has B-type stereospecificity with respect to hydride transfer from NADH. Analysis of initial velocity patterns together with product and substrate inhibition patterns and comparison of primary deuterium isotope effects on the apparent kinetic parameters, (D)k(cat), (D)(k(cat)/K(NADH)), and (D)(k(cat)/K(fructose)), show that MDH has an ordered kinetic mechanism at pH 8.2 in which NADH adds before D-fructose, and D-mannitol and NAD are released in that order. Isomerization of E-NAD to a form which interacts with D-mannitol nonproductively or dissociation of NAD from the binary complex after isomerization is the slowest step (>/=110 s(-)(1)) in D-fructose reduction at pH 8.2. Release of NADH from E-NADH (32 s(-)(1)) is the major rate-limiting step in mannitol oxidation at this pH. At the pH optimum for D-fructose reduction (pH 7.0), the rate of hydride transfer contributes significantly to rate limitation of the catalytic cascade and the overall reaction. (D)(k(cat)/K(fructose)) decreases from 2.57 at pH 7.0 to a value of

Characterization of dTDP-4-dehydrorhamnose 3,5-epimerase and dTDP-4-dehydrorhamnose reductase, required for dTDP-L-rhamnose biosynthesis in Salmonella enterica serovar Typhimurium LT2.

The thymidine diphosphate-L-rhamnose biosynthesis pathway is required for assembly of surface glycoconjugates in a growing list of bacterial pathogens, making this pathway a potential therapeutic target. However, the terminal reactions have not been characterized. To complete assignment of the reactions, the four enzymes (RmlABCD) that constitute the pathway in Salmonella enterica serovar Typhimurium LT2 were overexpressed. The purified RmlC and D enzymes together catalyze the terminal two steps involving NAD(P)H-dependent formation of dTDP-L-rhamnose from dTDP-6-deoxy-D-xylo-4-hexulose. RmlC was assigned as the thymidine diphosphate-4-dehydrorhamnose 3,5-epimerase by showing its activity to be NAD(P)H-independent. Spectrofluorometric and radiolabeling experiments were used to demonstrate the ability of RmlC to catalyze the formation of dTDP-6-deoxy-L-lyxo-4-hexulose from dTDP-6-deoxy-D-xylo-4-hexulose. Under reaction conditions, RmlC converted approximately 3% of its substrate to product. RmlD was unequivocally identified as the thymidine diphosphate-4-dehydrorhamnose reductase. The reductase property of RmlD was shown by equilibrium analysis and its ability to enable efficient biosynthesis of dTDP-L-rhamnose, even in the presence of low amounts of dTDP-6-deoxy-L-lyxo-4-hexulose. Comparison of 23 known and predicted RmlD sequences identified several conserved amino acid residues, especially the serine-tyrosine-lysine catalytic triad, characteristic for members of the reductase/epimerase/dehydrogenase protein superfamily. In conclusion, RmlD is a novel member of this protein superfamily.