COFACTOR SPECIFICITY ENGINEERING OF STREPTOCOCCUS MUTANS NADH OXIDASE 2 FOR NAD(P)+ REGENERATION IN BIOCATALYTIC OXIDATIONS

Barbara Petschacher,Nicole Staunig,Monika Müller,Martin Schürmann,Daniel Mink,Stefaan De Wildeman,Karl Gruber,Anton Glieder

doi:10.5936/csbj.201402005

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 93R94H showed comparable high rates and low Km values for NADPH (kcat 20 s−1, Km 6 μM) and NADH (kcat 25 s−1, Km 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.

Highlights

Enzyme catalyzed oxidation reactions have gained increasingEnzyme based, electrochemical, chemical, and photochemical interest in biocatalysis recently, reflected by a number of excellent regeneration methods are known
Coupled substrate or coupled reviews on this topic published in the last years [ -3]. enzyme systems [4, 3, 4] constitute two possibilities for enzymatic
Oxidoreductases constitute an important group of biocatalysts as they NAD(P)+ recycling

Summary

Introduction

Enzyme catalyzed oxidation reactions have gained increasingEnzyme based, electrochemical, chemical, and photochemical interest in biocatalysis recently, reflected by a number of excellent regeneration methods are known. Oxidoreductases constitute an important group of biocatalysts as they NAD(P)+ recycling. In these reaction set-ups the cofactor is facilitate the widely used stereoselective reduction of regenerated via the reduction of a carbonyl group of a cosubstrate, aldehydes and ketones and the less well exploited oxidation of catalyzed either by the production enzyme itself (coupled substrate) alcohols and amines. Oxidoreductases catalyzed oxidations are [ 5] or by an added dehydrogenase (coupled enzyme; used for production of chiral alcohols and amines by deracemization glutamate dehydrogenase [ 6, 7], lactate dehydrogenase [ 8]). Oxidoreductases, especially aldo-keto-reductases and dehydrogenases, act on the substrate by the transfer of electrons from or to a cofactor, mostly the nicotinamide-based nucleotides NAD(H) and NADP(H). While for the regeneration of the reduced cofactors NADH and NADPH several systems (engineered formate dehydrogenase [7,8], phosphite dehydrogenase [9, 0], glucose dehydrogenase [ , 2] plus cosubstrate) are well established and widely used, universal regeneration systems for the oxidized forms NAD+ and NADP+ are less well developed

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