Structure-guided engineering of the coenzyme specificity of Pseudomonas fluorescens mannitol 2-dehydrogenase to enable efficient utilization of NAD(H) and NADP(H)

The structure of Pseudomonas fluorescens mannitol 2-dehydrogenase with bound NAD(+) leads to the suggestion that the carboxylate group of Asp(69) forms a bifurcated hydrogen bond with the 2' and 3' hydroxyl groups of the adenosine of NAD(+) and contributes to the 400-fold preference of the enzyme for NAD+ as compared to NADP(+). Accordingly, the enzyme with the Asp(69) -> Ala substitution was found to use NADP(H) almost as well as wild-type enzyme uses NAD(H). The Glu(68) -> Lys substitution was expected to enhance the electrostatic interaction of the enzyme with the 2'-phosphate of NADP(+). The Glu(68) -> Lys:Asp(69) -> Ala doubly mutated enzyme showed about a 10-fold preference for NADP(H) over NAD(H), accompanied by a small decrease in catalytic efficiency for NAD(H)-dependent reactions as compared to wild-type enzyme. (c) 2007 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.