CHARACTERIZATION OF A DINUCLEOTIDE-BINDING SITE IN MONOAMINE OXIDASE-B BY SITE-DIRECTED MUTAGENESIS

Monoamine oxidase B (MAO B), an integral protein of the outer mitochon drial membrane, catalyzes the oxidative deamination of neuroactive and vasoactive amines, The oxidation step is coupled to the reduction of an obligatory FAD cofactor, In this study, we have examined the role o f one amino acid (Glu(34)) in human MAO B that is thought to play a cr ucial role in binding to the 2'-hydroxy group of ribose in the AMP moi ety of FAD. Glu(34) is located within a region of the MAO B molecule o f high sequence identity to the dinucleotide-binding site in other fla voproteins, In MAO B, this region is postulated to consist of a beta(1 )-sheet-alpha-helix-beta(2)-sheet motif which culminates with a Glu at the C-terminal end of the second beta-sheet, We used site-directed mu tagenesis to convert Glu at position 34 to Asp, Gin, and Ala. The wild -type and mutant cDNAs were then transiently transfected into COS-7 ce lls and assayed for MAO B activity, All three variants exhibited a dra matic decrease in the enzymatic activity as compared to wild-type MAO B, and only the Asp variant retained any detectable activity, Our stud ies indicate that the Glu(34) residue in human MAO B is essential for catalysis, Whether Glu(34) is responsible only for alignment of the FA D for participation in the oxidation/reduction cycle or also for the i nitial binding of FAD to the apoenzyme remains to be determined. (C) 1 995 Academic Press, Inc.