IDENTIFICATION OF THE COVALENT FLAVIN ATTACHMENT SITE IN SARCOSINE OXIDASE

Sarcosine oxidase from Corynebacterium sp. P-I is a heterotetrameric e nzyme (alpha beta gamma delta), that contains two noncovalently bound coenzymes (FAD, NAD(+)) and covalently bound FMN [8 alpha-(N-3-histidy l)FMN] which is attached to the beta subunit. Chlumsky et al. [(1995) J. Biol. Chem. 270, 18252-18259] tentatively identified His175 as the covalent FMN attachment site in the beta subunit, based on an alignmen t of the sequence of C. sp. P-l beta subunit with a highly homologous flavin-containing peptide from another corynebacterial sarcosine oxida se (C. sp. U-96) To test this hypothesis, His175 in the C. sp, P-I bet a subunit was mutated to an alanine. Unexpectedly, the mutant enzyme w as found to contain 1 mol of covalently bound flavin and to exhibit ca talytic activity similar to wild-type enzyme. Covalent flavin-containi ng peptides were isolated from wild-type and mutant enzymes and analyz ed by electrospray mass spectrometry, The mass observed for the mutant peptide (1152.4 Da) matched that predicted for an FMN-containing hexa peptide, corresponding to residues 173-178 (1152.1 Da). In the mutant, this region (HDAVAW) contains a single histidine (His173) which must be the covalent flavin attachment site, The mass observed for the wild -type peptide (1218.6 Da) matched that predicted for an FMN-containing hexapeptide, also corresponding to residues 173-178 in the beta subun it (1218.2 Da). This region in the wild-type enzyme includes two histi dine residues (HDHVAW). Attempts to sequence the wild-type or mutant p eptides by automated Edman degradation were unsuccessful, Instead, the peptide sequences were investigated by collisional-activated dissocia tion (CAD) and tandem mass spectrometry, The CAD mass spectral data wi th the mutant peptide confirmed the sequence deduced based on the mass of the intact peptide. The CAD mass spectral results with the wild-ty pe peptide showed that FMN was covalently attached to the N-terminal h istidine in the hexapeptide, which corresponds to His173 in the beta s ubunit.