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.
Bubner
Patricia
P
Institute of Biotechnology and Biochemical Engineering, Graz University of Technology, Petersgasse 12, Graz, Austria.
Klimacek
Mario
M
Nidetzky
Bernd
B
D001483:Base Sequence; D003067:Coenzymes / Q000378:metabolism*; D017931:DNA Primers; D007700:Kinetics; D008354:Mannitol Dehydrogenases / Q000235:genetics / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D009243:NAD / Q000378:metabolism*; D009249:NADP / Q000378:metabolism*; D011487:Protein Conformation; D015202:Protein Engineering; D011551:Pseudomonas fluorescens / Q000201:enzymology*; D013379:Substrate Specificity
D003067:Coenzymes; D017931:DNA Primers; D009243:NAD; D009249:NADP; D008354:Mannitol Dehydrogenases