Identification of the covalent flavin attachment site in sarcosine oxidase

Lawrence J. Chlumsky, Annie Willie Sturgess, Edward Nieves, Marilyn Schuman Jorns

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Abstract

Sarcosine oxidase from Corynebacterium sp. P-1 is a heterotetrameric enzyme (αβχδ) that contains two noncovalently bound coenzymes (FAD, NAD+) and covalently bound FMN [8α-(N3-histidyl)FMN] which is attached to the β subunit. Chlumsky et al. [(1995) J. Biol. Chem. 270, 18252-18259] tentatively identified His 175 as the covalent FMN attachment site in the β subunit, based on an alignment of the sequence of C. sp. P-1 β subunit with a highly homologous flavin-containing peptide from another corynebacterial sarcosine oxidase (C. sp. U-96). To test this hypothesis, His175 in the C. sp. P-1 β subunit was mutated to an alanine. Unexpectedly, the mutant enzyme was found to contain 1 mol of covalently bound flavin and to exhibit catalytic activity similar to wild-type enzyme. Covalent flavin-containing peptides were isolated from wild-type and mutant enzymes and analyzed by electrospray mass spectrometry. The mass observed for the mutant peptide (1152.4 Da) matched that predicted for an FMN-containing hexapeptide, 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 β subunit (1218.2 Da). This region in the wild-type enzyme includes two histidine residues (HDHVAW). Attempts to sequence the wild-type or mutant peptides by automated Edman degradation were unsuccessful. Instead, the peptide sequences were investigated by collisional-activated dissociation (CAD) and tandem mass spectrometry. The CAD mass spectral data with the mutant peptide confirmed the sequence deduced based on the mass of the intact peptide. The CAD mass spectral results with the wild-type peptide showed that FMN was covalently attached to the N- terminal histidine in the hexapeptide, which corresponds to His173 in the β subunit.

Original languageEnglish (US)
Pages (from-to)2089-2095
Number of pages7
JournalBiochemistry
Volume37
Issue number8
DOIs
Publication statusPublished - Feb 24 1998

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ASJC Scopus subject areas

  • Biochemistry

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