Rational Engineering of a Flavoprotein Oxidase for Improved Direct Oxidation of Alcohols to Carboxylic Acids.

Mathias Pickl,Marco Fraaije,Christoph Winkler,Silvia Glueck,Kurt Faber

doi:10.3390/molecules22122205

Abstract

The oxidation of alcohols to the corresponding carbonyl or carboxyl compounds represents a convenient strategy for the selective introduction of electrophilic carbon centres into carbohydrate-based starting materials. The O2-dependent oxidation of prim-alcohols by flavin-containing alcohol oxidases often yields mixtures of aldehyde and carboxylic acid, which is due to “over-oxidation” of the aldehyde hydrate intermediate. In order to directly convert alcohols into carboxylic acids, rational engineering of 5-(hydroxymethyl)furfural oxidase was performed. In an attempt to improve the binding of the aldehyde hydrate in the active site to boost aldehyde-oxidase activity, two active-site residues were exchanged for hydrogen-bond-donating and -accepting amino acids. Enhanced over-oxidation was demonstrated and Michaelis–Menten kinetics were performed to corroborate these findings.

Highlights

Oxidases are prominent biocatalysts due to their ability to utilize molecular oxygen as oxidant [1,2].In contrast to alcohol dehydrogenases, they do not suffer from an equilibrium problem, as the utilization of O2 as electron acceptor leads to a practically irreversible reaction [1,3]
Our strategy to improve the oxidation of the aldehyde hydrate via protein engineering focuses on the introduction of hydrogen-bond-donating or -accepting amino acids that support stabilization of the gem-diol in the active site, bearing in mind that an additional H-bond adds ~2 kcal·mol−1 of
Two residues interacting with the aldehyde moiety of the substrate via hydrogen bonding by positioning of the benzylic hydrogen atom in close proximity of N5 were selected: Val465, positioned directly on top of N5 of FAD and Trp466, performing π-π stacking with the benzylic moiety of the isoalloxazine ring of FAD (Figure 1) [8,20]

Summary

Introduction

Oxidases are prominent biocatalysts due to their ability to utilize molecular oxygen as oxidant [1,2].In contrast to alcohol dehydrogenases, they do not suffer from an equilibrium problem, as the utilization of O2 as electron acceptor leads to a practically irreversible reaction [1,3]. Oxidases are prominent biocatalysts due to their ability to utilize molecular oxygen as oxidant [1,2]. MP688 (HMFO, EC: 1.1.3.37), a well expressing, stable flavoprotein, that is active on a variety of benzylic or allylic prim-alcohols was described [4], which was termed for its ability to oxidize 5-(hydroxymethyl)furfural [5]. The latter can be obtained from hexoses on a large scale, and its oxidation to 2,5-furandicarboxylic acid furnishes a bio-based monomer for PEF as a substitute for petroleum-derived PET [6].

Methods

Discussion

Conclusion