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Identification of the Covalent Flavin Attachment Site in Sarcosine Oxidase
Journal article   Peer reviewed

Identification of the Covalent Flavin Attachment Site in Sarcosine Oxidase

Lawrence J Chlumsky, Annie Willie Sturgess, Edward Nieves and Marilyn Schuman Jorns
Biochemistry (Easton), v 37(8), pp 2089-2095
24 Feb 1998
DOI: https://doi.org/10.1021/bi972705s
PMID: 9485355
Featured in Collection :   UN Sustainable Development Goals @ Drexel

Abstract

Sarcosine oxidase from Corynebacterium sp. P-1 is a heterotetrameric enzyme (αβγδ) that contains two noncovalently bound coenzymes (FAD, NAD+) and covalently bound FMN [8α-(N 3-histidyl)FMN] which is attached to the β subunit. Chlumsky et al. [(1995) J. Biol. Chem. 270, 18252−18259] tentatively identified His175 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.

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Collaboration types
Domestic collaboration
Web of Science research areas
Biochemistry & Molecular Biology
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