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On the role of Bronsted catalysis in Pseudomonas fluorescens mannitol 2-dehydrogenase.

ABSTRACT: X-ray structure of the Pseudomonas fluorescens mannitol 2-dehydrogenase ternary complex with NAD+ and D-mannitol suggests that Lys-295 provides catalytic base assistance to secondary alcohol group oxidation. We have replaced Lys-295 by site-directed mutagenesis with alanine or methionine and evaluated the catalytic significance of side-chain substitution by kinetic analysis of restoration of activity with external amines, and from pH and solvent isotope effects on the reaction catalysed by K295A (Lys-295-->Ala mutant). K295A and K295M (Lys-295-->Met mutants) show 3x10(4)- and 2x10(6)-fold lower turnover numbers respectively for D-mannitol oxidation (kcatO) at pH 10.0 than the wild-type. The second-order rate constant for non-covalent rescue of activity (kB) by free methylamine base is 31 M(-1) x s(-1) for K295A, but only 0.021 M(-1) x s(-1) for K295M. A Brønsted relationship of log kB (corrected for molecular size effects) and pKa of the external amine is linear (slope beta=0.66+/-0.16; r2=0.99) for K295A-catalysed D-mannitol oxidation at pH 10.0. The kcatO values of K295A in H2O and 2H2O are linearly dependent on [OL-] in the pL range 7.5-10.5 (where L is 1H or 2H). The solvent isotope effect on kcatO is 0.69. The time course of D-fructose reduction by K295A at pH 8.2 displays a pre-steady-state burst of NADH consumption. These data support a mechanism in which the epsilon -NH2 group of Lys-295 participates in an obligatory pH-dependent, pre-catalytic equilibrium which may control alcohol/alkoxide equilibration of enzyme-bound D-mannitol and activates the C2 atom for subsequent catalytic oxidation by NAD+.

SUBMITTER: Klimacek M

PROVIDER: S-EPMC1223657 | biostudies-other | 2003 Oct

REPOSITORIES: biostudies-other

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Project description:Directional preference in catalysis is often used to distinguish alcohol dehydrogenases from carbonyl reductases. However, the mechanistic basis underpinning this discrimination is weak. In mannitol 2-dehydrogenase from Pseudomonas fluorescens, stabilization of (partial) negative charge on the substrate oxyanion by the side chains of Asn-191 and Asn-300 is a key feature of catalysis in the direction of alcohol oxidation. We have disrupted this ability through individual and combined substitutions of the two asparagines by aspartic acid. Kinetic data and their thermodynamic analysis show that the internal equilibrium of enzyme-NADH-fructose and enzyme-NAD(+)-mannitol (K(int)) was altered dramatically (10(4)- to 10(5)-fold) from being balanced in the wild-type enzyme (K(int) ? 3) to favoring enzyme-NAD(+)-mannitol in the single site mutants, N191D and N300D. The change in K(int) reflects a selective slowing down of the mannitol oxidation rate, resulting because Asn --> Asp replacement (i) disfavors partial abstraction of alcohol proton by Lys-295 in a step preceding catalytic hydride transfer, and (ii) causes stabilization of a nonproductive enzyme-NAD(+)-mannitol complex. N191D and N300D appear to lose fructose binding affinity due to deprotonation of the respective Asp above apparent pK values of 5.3 ± 0.1 and 6.3 ± 0.2, respectively. The mutant incorporating both Asn-->Asp substitutions behaved as a slow "fructose reductase" at pH 5.2, lacking measurable activity for mannitol oxidation in the pH range 6.8-10. A mechanism is suggested in which polarization of the substrate carbonyl by a doubly protonated diad of Asp and Lys-295 facilitates NADH-dependent reduction of fructose by N191D and N300D under optimum pH conditions. Creation of an effectively "one-way" reductase by active-site redesign of a parent dehydrogenase has not been previously reported and holds promise in the development of carbonyl reductases for application in organic synthesis.

Project description:Many serine hydrolases catalyze perhydrolysis, the reversible formation of peracids from carboxylic acids and hydrogen peroxide. Recently, we showed that a single amino acid substitution in the alcohol binding pocket, L29P, in Pseudomonas fluorescens (SIK WI) aryl esterase (PFE) increased the specificity constant of PFE for peracetic acid formation >100-fold [Bernhardt et al. (2005) Angew. Chem., Int. Ed. 44, 2742]. In this paper, we extend this work to address the three following questions. First, what is the molecular basis of the increase in perhydrolysis activity? We previously proposed that the L29P substitution creates a hydrogen bond between the enzyme and hydrogen peroxide in the transition state. Here we report two X-ray structures of L29P PFE that support this proposal. Both structures show a main chain carbonyl oxygen closer to the active site serine as expected. One structure further shows acetate in the active site in an orientation consistent with reaction by an acyl-enzyme mechanism. We also detected an acyl-enzyme intermediate in the hydrolysis of epsilon-caprolactone by mass spectrometry. Second, can we further increase perhydrolysis activity? We discovered that the reverse reaction, hydrolysis of peracetic acid to acetic acid and hydrogen peroxide, occurs at nearly the diffusion limited rate. Since the reverse reaction cannot increase further, neither can the forward reaction. Consistent with this prediction, two variants with additional amino acid substitutions showed 2-fold higher k(cat), but K(m) also increased so the specificity constant, k(cat)/K(m), remained similar. Third, how does the L29P substitution change the esterase activity? Ester hydrolysis decreased for most esters (75-fold for ethyl acetate) but not for methyl esters. In contrast, L29P PFE catalyzed hydrolysis of epsilon-caprolactone five times more efficiently than wild-type PFE. Molecular modeling suggests that moving the carbonyl group closer to the active site blocks access for larger alcohol moieties but binds epsilon-caprolactone more tightly. These results are consistent with the natural function of perhydrolases being either hydrolysis of peroxycarboxylic acids or hydrolysis of lactones.

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On the role of Bronsted catalysis in Pseudomonas fluorescens mannitol 2-dehydrogenase.

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