NDLI: Cofactor Specificity Engineering of Streptococcus mutans NADH Oxidase 2 for NAD(P)+ Regeneration in Biocatalytic Oxidations

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Cofactor Specificity Engineering of Streptococcus mutans NADH Oxidase 2 for NAD(P)+ Regeneration in Biocatalytic Oxidations

Content Provider	PubMed Central
Author	Barbara, Petschacher Nicole, Staunig Müller, Monika Martin, Schürmann Mink, Daniel Wildeman, Stefaan De Gruber, Karl Glieder, Anton
Copyright Year	2014
Abstract	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.
Related Links	http://dx.doi.org/10.5936/csbj.201402005
Starting Page	201402005
File Format	PDF
ISSN	20010370
e-ISSN	20010370
Journal	Computational and Structural Biotechnology Journal
Volume Number	9
Language	English
Publisher	Research Network of Computational and Structural Biotechnology (RNCSB) Organization
Publisher Date	2014-02-26
Access Restriction	Open
Rights Holder	Research Network of Computational and Structural Biotechnology (RNCSB) Organization
Subject Keyword	Research in Higher Education
Content Type	Text
Resource Type	Article
Subject	Genetics Structural Biology Biochemistry Biophysics Computer Science Applications Biotechnology

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Cofactor Specificity Engineering of Streptococcus mutans NADH Oxidase 2 for NAD(P)+ Regeneration in Biocatalytic Oxidations