Influence of flavin analogue structure on the catalytic activities and flavinylation reactions of recombinant human liver monoamine oxidases A and B

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 recombin ant human liver monoamine oxidases A and B (MAO A and B). Nineteen differen t riboflavin analogues were tested with MAO A and nine with MAO B. MAO expr ession 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 analogue s in the growth medium. Flavin analogues with a variety of seven and eight substitutions are found to be covalently incorporated and to confer catalyt ic activity, The selectivities of MAO A and MAO B for flavin analogue incor poration are found to be similar, although 8 alpha-methylation of the flavi n 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 boun d 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 incorporat ed into MAO A and do not support catalytic activity. A flavin peptide was i solated from MAO A containing 7-nor-7-bromo-FAD and was demonstrated to be covalently attached to Cys-406 by an 8 alpha-8-thioether linkage by sequenc e analysis and by matrix-assisted laser desorption ionization time of fligh t mass spectroscopy. MAO A partially purified from yeast grown on 8-nor-8-c hlororiboflavin exhibited an absorption spectrum indicating the covalent fl avin is an 8-nor-8-S-thioflavin, suggesting a nucleophilic displacement mec hanism that supports the quinone-methide mechanism previously suggested as a general mechanism for covalent flavin attachment.