engleski

Oxidative protein modification and enzyme inactivation: insights from a structure-function study of Trigonopsis variabilis D-amino acid oxidase

Oxidases constitute a structurally diverse group of enzymes that catalyze a wide range of chemical reactions using molecular oxygen as the electron acceptor. Their ability to couple the selective transformation of a substrate with the controlled reduction of dioxygen into hydrogen peroxide is of great interest in biocatalysis. Unfortunately, oxidases often fall short in their stabilities under conditions of process operation, and one of the primary sources of the loss of activity is oxidative protein modification. Little is known, at the molecular level, about how the structural heterogeneity induced by the chemical conversion of an oxidationsensitive side chain can affect enzyme function. The information would be vital to devise a targeted approach for improving enzyme performance, especially with regard to stability. This thesis reports on the oxidative modification of D-amino acid oxidase (DAO), a flavin-dependent enzyme from the yeast Trigonopsis variabilis which is widely known for its application in the industrial conversion of Cephalosporin C. Using electrospray tandem mass spectrometry in combination with an assortment of biochemical methods, cysteine 108 was identified as the main site undergoing oxidation in the enzyme, and its conversion into a stable cysteine sulfinic in vivo was corroborated by oxidative modification studies on purified wild-type oxidase as well as two mutants thereof in which cysteine 108 was replaced by a serine and an aspartate. Functional consequences of chemical and genetic "mutations" of cysteine 108 were determined. Using a mechanistic model of the thermal inactivation of DAO which is composed of two parallel paths of denaturation – partial unfolding and cofactor dissociation – it was possible to explain changes in overall stability caused by modifications of cysteine 108 with altered rates of cofactor release. Modification of surface-exposed cysteines or mutation of cysteine 108 are proposed as novel approaches for the stabilization of DAO.

Oxidative Protein Modification; Enzyme Inactivation; Structure-Function Relationship; (D-amino acid) oxidase

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