Design and properties of human D-amino acid oxidase with covalently attached flavin.

An "artificial flavinylation" approach was developed to replace a native noncovalent flavin prosthetic group with a covalently attached flavin analogue in recombinant human d-amino acid oxidase. The protein residue Gly-281 was replaced with Cys by site-directed mutagenesis, followed by reaction between mutated apoenzyme and the thiol-reactive flavin analogue, 8-methylsulfonyl FAD. The stoichiometric process of flavin attachment was accompanied by gain in enzymatic activity, reaching up to 26% activity of the recombinant native enzyme. The steady-state kinetic data together with the results of limited proteolysis and benzoate-binding studies suggest that, although mutation perturbs protein structural and catalytic properties, the flavinylation alone does not have any negative impact. We conclude that, despite the implemented restraints on its mobility, the covalently attached flavin is properly positioned within the protein active site and acts efficiently during d-amino acid oxidase catalytic turnover.