Structure of the flavocoenzyme of two homologous amine oxidases: Monomericsarcosine oxidase and N-methyltryptophan oxidase

Monomeric sarcosine oxidase (MSOX) and N-methyltryptophan oxidase (MTOX) ar e homologous enzymes that catalyze the oxidative demethylation of sarcosine (N-methylglycine) and N-methyl-L-tryptophan, respectively. MSOX is induced in various bacteria upon growth on sarcosine. MTOX is an E, coli enzyme of unknown metabolic function, Both enzymes contain covalently bound flavin. The covalent flavin is at the FAD level as judged by electrospray mass spec trometry, The data provide the first evidence that MTOX is a flavoprotein. The following observations indicate that 8 alpha-(S-cysteinyl)FAD is the co valent flavin in MSOX from Bacillus sp. B-0618 and MTOX. FMN-containing pep tides, prepared by digestion of MSOX or MTOX with trypsin, chymotrypsin, an d phosphodiesterase, exhibited absorption and fluorescence properties chara cteristic of an 8 alpha-(S-cysteinyl)flavin and could be bound to apo-flavo doxin. The thioether link in the FMN-containing peptides was converted to t he sulfone by performic acid oxidation, as judged by characteristic absorba nce changes and an increase in flavin fluorescence. The sulfone underwent a predicted reductive cleavage reaction upon treatment with dithionite, rele asing unmodified FMN, Cys315 was identified as the covalent FAD attachment site in MSOX from B. sp. B-0618, as judged by the sequence obtained for a f lavin-containing tryptic peptide (GAVCMYT). Cys315 aligns with a conserved cysteine in MSOX from other bacteria, MTOX (Cys308) and pipecolate oxidase, a homologous mammalian enzyme known to contain covalently bound flavin. Th ere is only one conserved cysteine found among these enzymes, suggesting th at Cys308 is the covalent flavin attachment site in MTOX.