Abstract
The VAO flavoprotein family consists mostly of oxidoreductases harboring a covalently linked flavin cofactor. The linkage can be either monocovalent at position 8 with a histidine or tyrosine or bicovalent at position 8 with a histidine and at position 6 with a cysteine. Bicovalently bound flavoproteins show a preference for bulkier substrates such as oligosaccharides or secondary metabolites. The genome of the thermophilic fungus Myceliophthora thermophila C1 was found to be rich in genes encoding putative covalent VAO-type flavoproteins. Enzymes from this fungus have the advantage of being rather thermostable and homologous overexpression in M. thermophila C1 is feasible. Recently we discovered a new and VAO-type carbohydrate oxidase from this fungus: xylooligosaccharide oxidase. In this study, two other putative VAO-type oxidases, protein sequence XP_003663615 (MtVAO615) and XP_003665713 (MtVAO713), were expressed in M. thermophila C1, purified and characterized. Enzyme MtVAO615 was found to contain a bicovalently bound FAD, while enzyme MtVAO713 contained a monocovalent histidyl-bound FAD. The crystal structures of both proteins were obtained which revealed atypical active site architectures. It could be experimentally verified that both proteins, when reduced, rapidly react with molecular oxygen, a hallmark of flavoprotein oxidases. A large panel of alcohols, including carbohydrates, steroids and secondary alcohols were tested as potential substrates. For enzyme MtVAO713 low oxidase activity was discovered towards ricinoleic acid.
Highlights
Oxidases are enzymes capable of performing selective oxidative reactions using molecular oxygen as electron acceptor
We recently reported on the discovery and the elucidated crystal structure of a new vanillyl alcohol oxidase (VAO)-type oxidase: xylooligosaccharide oxidase (XylO) from M. thermophila XylO has a similar active site when compared to previously determined structures of other known VAO-type carbohydrate oxidases such as glucooligosaccharide oxidase from Acremonium strictum (GOOX) and lactose oxidase from
Upon multiple sequence alignment of the recovered protein sequences with sequences from VAO-type flavoproteins for which a crystal structure was elucidated, a phylogenetic tree was built (Figure 1). Given their high similarity with MtVAO713, two additional sequences were included in the tree: an alcohol oxidase from Hypomyces subiculosus and ecdysteroid-22-oxidase from Nomuraea rileyi
Summary
Oxidases are enzymes capable of performing selective oxidative reactions using molecular oxygen as electron acceptor. They represent valuable and cost-effective biotechnological tools for industrial applications since they do not require expensive coenzymes but just molecular oxygen. Oxidases typically employ a copper ion or a flavin as cofactor. Known examples of the former are galactose oxidase and laccases. The flavin-containing group of oxidases is more abundant. Oxidases in this category contain mostly a flavin adenine dinucleotide (FAD) as prosthetic group with a minority containing flavin mononucleotide (FMN). Based on sequence homology and the available structural information, six families of flavoprotein oxidases have been identified [1]
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