Cofactor Specificity Engineering of Streptococcus mutans NADH Oxidase 2 for NAD(P)(+) Regeneration in Biocatalytic Oxidations.
Soluble water-forming NAD(P)H oxidases constitute a promising NAD(P)(+) regeneration method as they only need oxygen as cosubstrate and produce water as sole byproduct. Moreover, the thermodynamic equilibrium of O2 reduction is a valuable driving force for mostly energetically unfavorable biocatalytic oxidations. Here, we present the generation of an NAD(P)H oxidase with high activity for both cofactors, NADH and NADPH. Starting from the strictly NADH specific water-forming Streptococcus mutans NADH oxidase 2 several rationally designed cofactor binding site mutants were created and kinetic values for NADH and NADPH conversion were determined. Double mutant 193R194H showed comparable high rates and low K m values for NADPH (k cat 20 s(-1), K m 6 µM) and NADH (k cat 25 s(-1), K m 9 µM) with retention of 70% of wild type activity towards NADH. Moreover, by screening of a SeSaM library S. mutans NADH oxidase 2 variants showing predominantly NADPH activity were found, giving further insight into cofactor binding site architecture. Applicability for cofactor regeneration is shown for coupling with alcohol dehydrogenase from Sphyngobium yanoikuyae for 2-heptanone production.
Austrian Centre of Industrial Biotechnology GmbH, c/o Institute of Molecular Biotechnology, Graz University of Technology, Petersgasse 14, 8010 Graz, Austria.
Barbara
B
Petschacher
Austrian Centre of Industrial Biotechnology GmbH, c/o Institute of Molecular Biosciences, University Graz, Humboldtstrasse 50/3, 8010 Graz, Austria.
Nicole
N
Staunig
DSM Innovative Synthesis B.V., P.O. Box 18, 6160 MD Geleen, Netherlands.
Monika
M
Müller
DSM Innovative Synthesis B.V., P.O. Box 18, 6160 MD Geleen, Netherlands.
Martin
M
Schürmann
DSM Innovative Synthesis B.V., P.O. Box 18, 6160 MD Geleen, Netherlands.
Daniel
D
Mink
DSM Innovative Synthesis B.V., P.O. Box 18, 6160 MD Geleen, Netherlands.
Stefaan
S
De Wildeman
Austrian Centre of Industrial Biotechnology GmbH, c/o Institute of Molecular Biosciences, University Graz, Humboldtstrasse 50/3, 8010 Graz, Austria.
Karl
K
Gruber
Austrian Centre of Industrial Biotechnology GmbH, c/o Institute of Molecular Biotechnology, Graz University of Technology, Petersgasse 14, 8010 Graz, Austria.
Anton
A
Glieder
2-heptanol oxidation; NADPH oxidase; NADPH recycling; coenzyme selectivity; cofactor regeneration; site-directed mutagenesis