A Specific Mannitol Dehydrogenase from Lactobacillus brevis

Martínez, Germán David Mendoza; Barker, H. A.; Horecker, B.L.

doi:10.1016/s0021-9258(18)81106-1

Cited by 70 publications

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“…It can be utilized as a carbon source for growth and energy, a carbon storage compound, a free radical scavenger, or a compatible solute providing protection against various forms of stress (). The initial step in the catabolism of d -mannitol involves oxidations at the C-1 ( , ) and C-2 ( − ) positions, catalyzed by NAD(P)-dependent dehydrogenases. The reaction catalyzed by mannitol 2-dehydrogenase (MDH, EC 1.1.1.67) is At present, there is little known about the structure−function relationships of MDH, and the kinetic and chemical mechanisms of MDH have not been studied, except for an early kinetic study with the MDH from the fungus Absidia glauca ().…”

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Kinetic Study of the Catalytic Mechanism of Mannitol Dehydrogenase from Pseudomonas fluorescens

1999

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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

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