PhD ceremony: D.P.H.M. Heuts, 14.45 uur, Academiegebouw, Broerstraat 5, Groningen
Thesis: Structural and kinetic studies on novel covalent flavoprotein oxidases: alditol oxidase and chitooligosaccharide oxidase
Promotor(s): prof. M.W. Fraaije, prof. D.B. Janssen
Faculty: Mathematics and Natural Sciences
Flavoprotein oxidases are becoming more interesting to the fine-chemical and pharmaceutical industries. The focus of this PhD research was to the search for novel carbohydrate oxidases. Two putative covalent flavin-containing carbohydrate oxidases were selected using the genome database mining approach. This thesis presents the kinetic and structural analyses alditol oxidase (AldO) from the bacterium Streptomyces coelicolor and chitooligosaccharide oxidase (ChitO) from the fungus Fusarium graminearum.
AldO catalyzes the selective oxidation of primary alcohols to their corresponding aldehydes. The best substrate for AldO is xylitol yielding D-xylose as sole product. The results of a detailed kinetic analysis corresponded to a ternary complex mechanism to be operative. High-resolution structures of AldO were obtained with substrate bound, an FAD-sulfite adduct and as native protein. This confirmed that AldO contains an FAD cofactor that is covalently linked to His46. The substrate bound structures reveal why only one primary hydroxyl moiety on the substrate is oxidized. AldO may be involved in sorbitol or xylitol catabolism, or in the biosynthesis of secondary metabolites.
ChitO is active with N-acetylated oligosaccharides, so-called chitooligosaccharides. One amino acid residue (Gln268) was found to be responsible for this substrate specificity. Based on sequential-, spectral- and mutagenic analysis it was found that ChitO contains a bi-covalently linked flavin cofactor. The double covalent bond is not only required for redox tuning but also for positioning the FAD cofactor in the active site. The idea behind this is that the bi-covalent binding of FAD may be required for the formation of a productive Michaelis-complex.
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