MUTAGENESIS AT A HIGHLY CONSERVED TYROSINE IN MONOAMINE-OXIDASE-B AFFECTS FAD INCORPORATION AND CATALYTIC ACTIVITY

Monoamine oxidase B (MAO B), an integral protein of the outer mitochon drial membrane, catalyzes the oxidative deamination of various neuroac tive and vasoactive amines. A covalently bound FAD cofactor at Cys-397 of human MAO B is required for the oxidation of the amine substrates. In addition to the covalent binding site, MAO B also contains a nonco valent FAD binding region (residues 6-34) known as the dinucleotide bi nding motif. Previously, we have shown that Glu-34 is required for cat alytic activity, presumably by forming a hydrogen bond between the car boxylate group of glutamate and the 2'-hydroxyl group of ribose in the AMP moiety of FAD. In this work, we have identified a third FAD bindi ng site in MAO B (residues 39-46) by sequence comparisons to other fla voenzymes. The conserved sequence contains a tyrosine residue (Tyr-44) which, based on the X-ray crystal structure of ferredoxin-NADP(+) red uctase, is postulated to participate in FAD binding through van der Wa als contact with the isoalloxazine ring and a hydrogen bond to the 3'- hydroxy of the ribityl moiety. To test the postulated role of this tyr osine residue, site-directed mutants that encode substitutions at Tyr- 44 were prepared and expressed in mammalian COS-7 cells. Variant MAO B enzymes were then characterized with respect to enzymatic activity an d [C-14]FAD incorporation. Substitution of tyrosine with phenylalanine had no effect on MAO B activity or the level of [C-14]FAD incorporati on compared to the wild-type enzyme, indicating that the hydroxyl grou p of the tyrosine residue was not essential at residue 44. Substitutio n of tyrosine with serine or alanine, however, which do not have an ar omatic ring, resulted in a dramatic decrease in enzymatic activity and FAD incorporation, We conclude that the aromatic ring of the tyrosine residue at position 44 is required for FAD binding and catalytic acti vity of MAO B.