Evolution and engineering of pyranose oxidoreductases for modified oxygen reactivity
Abstract
Reactions of flavoenzyme oxidoreductases consist of a reductive half-reaction, in which the substrate reduces the flavin, and an oxidative half-reaction, in which flavin is re-oxidized by an electron acceptor. Flavin-dependent oxidases use dioxygen as electron acceptor, producing hydrogen peroxide, whereas dehydrogenases and electron transferases react either very slowly or not at all with oxygen, and utilize alternative electron acceptors such as various (substituted) quinones or redox-proteins. Despite intensive research, the chemical and structural basis of oxygen reactivity remains largely undetermined, and specific binding sites for oxygen or alternative electron acceptors could be found. Several amino acid residues were shown to influence oxygen reactivity substantially in various proteins of different structural families, but no structural determinants governing whether an enzyme reacts as an oxidase or a dehydrogenase could be estabished. Pyranose 2-oxidase and pyranose dehydrogenase are two flavin-dependent sugar oxidoreductases from mainly white-rot and litter-decomposing basidiomycetes, respectively, which catalyze the regioselective oxidation of various aldopyranoses at the C-2 position (and sometimes at C-3). P2Ox is a homotetrameric protein and acts as an oxidase, PDH is a monomeric glycoprotein and is dependant on alternative acceptors such as 1,4-benzoquinone, the ferricenium ion and ferricyanide. Both belong to the GMC-family of flavin-dependant enzymes, and their active sites share a high degree of similarity. Genes encoding both enzymes were isolated in our laboratory, and heterologous expression was established. We propose to use an approach combining methods of directed evolution as well as semi-rational protein engineering, in order to change, and to identify amino acids influencing, the oxygen reactivity. Mini-libraries of enzyme variants will be produced by saturation mutagenesis of amino acids surrounding the active site, as well as libraries of randomly mutated p2ox- and pdh-genes, and screened in a parallel assay for changes in catalytic activity with oxygen and alternative electron acceptors. Mutations that show an effect will be biochemically characterized in detail. The similarity of the two enzymes active sites will allow comparisons of effects of certain amino acid exchanges, as well as the confirmation of these effects by introduction of identical and complementary alterations in both enzymes. Besides increasing basic knowledge about the reaction chemistry of enzymes from the GMC family of flavin-dependent enzymes, this research has some applicational relevance as well, as it would, if successful, allow to, e.g., eliminate the detrimental hydrogen peroxide production by P2Ox, or utilize the more flexible PDH in biocatalysis without redox mediator regenerations.
keywords Flavoproteins Carbohydrate oxidoreductases Electron acceptors Enzyme engineering Directed evolution
Publikationen
Project staff
Clemens Karl Peterbauer
Assoc. Prof. Dr. Clemens Karl Peterbauer
clemens.peterbauer@boku.ac.at
Tel: +43 1 47654-75212
Project Leader
01.03.2010 - 31.05.2013