The FAD binding sites of human liver monoamine oxidases A and B: investigation of the role of flavin ribityl side chain hydroxyl groups in the covalent flavinylation reaction and catalytic activities

The role of ribityl side chain hydroxyl groups of the flavin moiety in the covalent flavinylation reaction and catalytic activities of recombinant hum an liver monoamine oxidases (MAO) A and B have been investigated using the riboflavin analogue: N(10)-omega-hydroxypentyl-isoalloxazine. Using a rib5 disrupted strain of Saccharomyces cerevisiae which is auxotrophic for ribof lavin, MAO A and MAO B were expressed separately under control of a galacto se inducible GAL10/ CYC1 promoter in the presence of N(10)-omega-hydroxypen tyl-isoalloxazine as the only available riboflavin analogue. Analysis of mi tochondrial membrane proteins shows both enzymes to be expressed at levels comparable to those cultures grown on riboflavin and to contain covalently bound flavin. Catalytic activities, as monitored by kynuramine oxidation, a re equivalent to (MAO A) 2-fold greater (MAO B) than control preparations e xpressed in the presence of riboflavin. Although N(10)-omega-hydroxypentyl- isoalloxazine is unable to support growth of riboflavin auxotrophic S. cere visiae, it is converted to the FMN level by yeast cell free extracts. The F MN form of the analogue is converted to the FAD level by the yeast FAD synt hetase, as shown by expression of the recombinant enzyme in Escherichia col i. These data show that the ribityl hydroxyl groups of the FAD moiety are n ot required for covalent flavinylation or catalytic activities of monoamine oxidases A and B. This is in contrast to the suggestion based on mutagenes is studies that an interaction between the 3'-hydroxyl group of the flavin and the beta-carbonyl of Asp(227) is required for the covalent flavinylatio n reaction of MAO B (Zhou et al., J. Biol. Chem, 273 (1998) 14862-14868), ( C) 2000 Elsevier Science B.V. All rights reserved.