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J Biol Chem, Vol. 274, Issue 33, 23515-23525, August 13, 1999

Influence of Flavin Analogue Structure on the Catalytic Activities and Flavinylation Reactions of Recombinant Human Liver Monoamine Oxidases A and B

From the Departments of Biochemistry and Chemistry, Emory University School of Medicine, Atlanta, Georgia 30322

Two riboflavin-deficient (rib5) Saccharomyces cerevisiae expression systems have been developed to investigate the influence of riboflavin structural alterations on the covalent flavinylation reaction and activity of recombinant human liver monoamine oxidases A and B (MAO A and B). Nineteen different riboflavin analogues were tested with MAO A and nine with MAO B. MAO expression and flavinylation were determined immunochemically with antisera to MAO and an anti-flavin antisera. Expression levels of both MAO A and B are invariant with the presence or absence of riboflavin or riboflavin analogues in the growth medium. Flavin analogues with a variety of seven and eight substitutions are found to be covalently incorporated and to confer catalytic activity. The selectivities of MAO A and MAO B for flavin analogue incorporation are found to be similar, although 8-methylation of the flavin resulted in a higher level of catalytic activity for MAO B than for MAO A. N(3)-Methylriboflavin and 8-nor-8-aminoriboflavin are not covalently bound as they are not converted to their respective FAD forms by yeast. 5-Carba-5-deazaflavin and 7,8-nor-7-chlororiboflavin are not covalently incorporated into MAO A and do not support catalytic activity. A flavin peptide was isolated from MAO A containing 7-nor-7-bromo-FAD and was demonstrated to be covalently attached to Cys-406 by an 8-S-thioether linkage by sequence analysis and by matrix-assisted laser desorption ionization time of flight mass spectroscopy. MAO A partially purified from yeast grown on 8-nor-8-chlororiboflavin exhibited an absorption spectrum indicating the covalent flavin is an 8-nor-8-S-thioflavin, suggesting a nucleophilic displacement mechanism that supports the quinone-methide mechanism previously suggested as a general mechanism for covalent flavin attachment.

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