Engineering of Pyranose Dehydrogenase for Increased Oxygen Reactivity

Abstract

Pyranose dehydrogenase (PDH), a member of the GMC family of flavoproteins, shows a very broad sugar substrate specificity but is limited to a narrow range of electron acceptors and reacts extremely slowly with dioxygen as acceptor. The use of substituted quinones or (organo)metals as electron acceptors is undesirable for many production processes, especially of food ingredients. To improve the oxygen reactivity, site-saturation mutagenesis libraries of twelve amino acids around the active site of Agaricus meleagris PDH were expressed in Saccharomyces cerevisiae. We established high-throughput screening assays for oxygen reactivity and standard dehydrogenase activity using an indirect Amplex Red/horseradish peroxidase and a DCIP/D-glucose based approach. The low number of active clones confirmed the catalytic role of H512 and H556. Only one position was found to display increased oxygen reactivity. Histidine 103, carrying the covalently linked FAD cofactor in the wild-type, was substituted by tyrosine, phenylalanine, tryptophan and methionine. Variant H103Y was produced in Pichia pastoris and characterized and revealed a five-fold increase of the oxygen reactivity.

Agaricus meleagris pyranose dehydrogenase: Influence of covalent FAD linkage on catalysis and stability

“…A fivefold increase in oxygen reactivity was recently reported for this variant [16]. In VAO, the same effect was observed.…”

“…The catalytic efficiencies towards sugar substrates and electron acceptors decreased substantially [16]. However, these findings could not be explained satisfactorily.…”

Engineering of pyranose 2-oxidase for modified oxygen reactivity

“…Reduced oxygen reactivity is a desirable property for some applications because of reduced The reactivity of flavoproteins with oxygen is of considerable scientific interest, and the determinants of oxygen activation and reactivity are the subject of numerous studies [39][40][41][42][43][44]. The consensus is that there is no single parameter (like spatial accessibility, charge, hydrophobicity, vicinity of particular amino acid residues etc.)…”

“…Since oxygen-utilizing enzymes appeared later in evolution than dehydrogenases, which already existed in an atmosphere devoid of molecular oxygen, this is not surprising. We recently pursued a similar objective in the catalytically related pyranose dehydrogenase, an enzyme that reacts extremely slowly with oxygen, namely to substantially increase oxidase activity, either with unaltered or at the expense of dehydrogenase activity [39]. In this work, which followed an essentially identical approach of substituting amino acid residues in proximity of the flavin C(4a) by site-saturation mutagenesis, we identified only one residue that influenced oxidase activity to a measurable degree: His103, carrying the covalently attached flavin cofactor, with the observed increase in oxygen reactivity most likely due to changes in the redox potential of the cofactor [39].…”

“…Channels leading from the surface to the active site may affect oxidase reactivity [37,38]. Altering the oxygen reactivity is of considerable interest as shown in recent reviews [15,28] and works of Krondorfer et al [39], Sygmund et al [40], Leferink et al [41], and others [42][43][44].…”