Abstract
By inspection of the predicted proteome of the fungus Myceliophthora thermophila C1 for vanillyl-alcohol oxidase (VAO)-type flavoprotein oxidases, a putative oligosaccharide oxidase was identified. By homologous expression and subsequent purification, the respective protein could be obtained. The protein was found to contain a bicovalently bound FAD cofactor. By screening a large number of carbohydrates, several mono- and oligosaccharides could be identified as substrates. The enzyme exhibits a strong substrate preference toward xylooligosaccharides; hence it is named xylooligosaccharide oxidase (XylO). Chemical analyses of the product formed upon oxidation of xylobiose revealed that the oxidation occurs at C1, yielding xylobionate as product. By elucidation of several XylO crystal structures (in complex with a substrate mimic, xylose, and xylobiose), the residues that tune the unique substrate specificity and regioselectivity could be identified. The discovery of this novel oligosaccharide oxidase reveals that the VAO-type flavoprotein family harbors oxidases tuned for specific oligosaccharides. The unique substrate profile of XylO hints at a role in the degradation of xylan-derived oligosaccharides by the fungus M. thermophila C1.
Highlights
By inspection of the predicted proteome of the fungus Myceliophthora thermophila C1 for vanillyl-alcohol oxidase (VAO)type flavoprotein oxidases, a putative oligosaccharide oxidase was identified
We report on the discovery, characterization, and crystal structures of a novel flavoprotein oxidase belonging to the VAO family, which is primarily active toward xylooligosaccharides (XOS): xylooligosaccharide oxidase (XylO) from the thermophilic fungus Myceliophthora thermophila C1
Homology Analysis—We have recently identified a stretch of conserved residues that correlates with the presence of a bicovalently bound FAD in flavoprotein sequences that belong to the VAO flavoprotein family [12]
Summary
GluO, glucooligosaccharide oxidase; XylO, xylooligosaccharide oxidase; VAO, vanillyl alcohol oxidase; LaO, lactose oxidase; ChitO, chitooligosaccharide oxidase; XOS, xylooligosaccharide(s); TMS, trimethylsilyl; HMBC, heteronuclear multiple bond correlation; HSQC, heteronuclear single quantum coherence; RMSD, root mean square deviation; FAD domain, FAD binding domain; S domain, substrate binding domain. The nature and positioning of a number of residues that form the carbohydrate binding groove dictate which oligosaccharides can bind in such a way that the flavin cofactor is able to oxidize the substrate The role of these residues in substrate acceptance was clearly demonstrated by engineering ChitO: by replacing three residues, an efficient lactose oxidase could be generated from ChitO [11]. This shows that the nature of the amino acids lining the carbohydrate binding groove in oligosaccharide oxidases determines the substrate specificity. The results show that XylO can be used for generating XOS-based aldonic acids
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