Publications

• Identification of the covalent flavin attachment site in sarcosine oxidase.

  Chlumsky L.J., Sturgess A.W., Nieves E., Jorns M.S.

  Sarcosine oxidase from Corynebacterium sp. P-1 is a heterotetrameric enzyme (alphabetagammadelta) that contains two noncovalently bound coenzymes (FAD, NAD+) and covalently bound FMN [8alpha-(N3-histidyl)FMN] which is attached to the beta subunit. Chlumsky et al. [(1995) J. Biol. Chem. 270, 18252- ... >> More

  Sarcosine oxidase from Corynebacterium sp. P-1 is a heterotetrameric enzyme (alphabetagammadelta) that contains two noncovalently bound coenzymes (FAD, NAD+) and covalently bound FMN [8alpha-(N3-histidyl)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 alignment of the sequence of C. sp. P-1 beta 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 beta 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 beta 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 beta subunit. << Less

  Biochemistry 37:2089-2095(1998) [PubMed] [EuropePMC]

• Folate utilization by monomeric versus heterotetrameric sarcosine oxidases.

  Wagner M.A., Schuman Jorns M.

  There are two types of bacterial sarcosine oxidases. The heterotetrameric enzymes contain subunits ranging in size from about 10 to 100 kDa, noncovalently bound FAD and NAD+, and covalently bound FMN attached to the beta subunit (42-45 kDa). Monomeric sarcosine oxidases are similar in size to the ... >> More

  There are two types of bacterial sarcosine oxidases. The heterotetrameric enzymes contain subunits ranging in size from about 10 to 100 kDa, noncovalently bound FAD and NAD+, and covalently bound FMN attached to the beta subunit (42-45 kDa). Monomeric sarcosine oxidases are similar in size to the beta subunit in the heterotetramers and contain covalently bound FAD. Formaldehyde formation during sarcosine oxidation by several heterotetrameric sarcosine oxidases was suppressed in the presence of 50 microM [6S]-tetrahydrofolate, accompanied by a 25-50% increase in the rate of sarcosine oxidation. In contrast, [6S]-tetrahydrofolate caused only a modest decrease in the rate of formaldehyde production with monomeric sarcosine oxidases (approximately 25%), an effect which was virtually entirely attributable to an accompanying decrease in the rate of sarcosine oxidation. In the presence of 100 microM [6R,S]-tetrahydropteroyltriglutamate [H4Pte(Glu)3], the heterotetrameric enzymes catalyzed the formation of 5,10-methylenetetrahydropteroyltriglutamate [5,10-CH2-H4Pte(Glu)3] at a rate which was 35-60% faster than the rate of sarcosine oxidation in the absence of folate. An apparent Km value of 3.1 microM was estimated for [6S]-H4Pte(Glu)3 with the heterotetrameric corynebacterial sarcosine oxidase. In contrast, slow formation of 5,10-CH2-H4Pte(glu)3 was detected during sarcosine oxidation with monomeric sarcosine oxidases, attributable to the nonenzymatic reaction of free formaldehyde with H4Pte(Glu)3. The results show that only the heterotetrameric sarcosine oxidases can use tetrahydrofolates as substrates and, in this regard, they resemble mammalian sarcosine and dimethylglycine dehydrogenases. << Less

  Arch. Biochem. Biophys. 342:176-181(1997) [PubMed] [EuropePMC]

• Preparation and properties of recombinant corynebacterial sarcosine oxidase: evidence for posttranslational modification during turnover with sarcosine.

  Chlumsky L.J., Zhang L., Ramsey A.J., Jorns M.S.

  The genes encoding the four subunits of sarcosine oxidase from Corynebacterium sp. P-1 were isolated and overexpressed in a single step by using indicator plates to screen a genomic library for colonies that generated hydrogen peroxide in a sarcosine-dependent reaction. The genomic library was con ... >> More

  The genes encoding the four subunits of sarcosine oxidase from Corynebacterium sp. P-1 were isolated and overexpressed in a single step by using indicator plates to screen a genomic library for colonies that generated hydrogen peroxide in a sarcosine-dependent reaction. The genomic library was constructed by inserting size-fractionated genomic DNA, previously subjected to partial digestion by Sau3AI, into pBluescript II SK (+). At least 1.0 kb, but less than 4.0 kb, can be deleted from the 3' end of the original cornyebacterial insert (7.3 kb) without affecting sarcosine oxidase expression, consistent with the estimated 5.0-kb operon size. Recombinant sarcosine oxidase is isolated as a heterotetramer containing equimolar amounts of covalent and noncovalent flavin, identical to that observed for enzyme isolated from Corynebacterium sp. P-1. Despite its similar flavin content, recombinant enzyme exhibits significantly different spectral properties than enzyme from Corynebacterium sp. P-1 (values shown in parentheses) [epsilon 450 = 9.7 (12.7) mM-1 cm-1; A368/A450 = 1.0 (0.83); A280/A450 = 16.9 (12.2)]. This difference is due to the fact that about half of the covalent flavin in recombinant enzyme forms a reversible covalent 4a-adduct with a cysteine residue (lambda max = 383 nm; epsilon 383 = 7.3 mM-1 cm-1). The equilibrium is shifted in favor of adduct dissociation by oxidizing the cysteine residue with hydrogen peroxide or by alkylation with methyl methanethiosulfonate in a reaction that is fully reversible upon addition of excess dithiothreitol. The cysteine residue is also oxidized during aerobic turnover with sarcosine. Reaction of the cysteine residue with hydrogen peroxide (or a precursor) formed during turnover partially competes with the release of hydrogen peroxide into solution, as judged by the effect of catalase on this reaction. Although the same specific activity is observed for recombinant enzyme and enzyme from Corynebacterium sp. P-1, the recombinant enzyme exhibits a pronounced lag in an NADH peroxidase-coupled assay. The lag is eliminated by prior disruption of the 4a-thiolate adduct via reaction with hydrogen peroxide or methyl methanethiosulfonate. The results show that the 4a-thiolate adduct is an inactive form of sarcosine oxidase that can be activated by reaction with sarcosine in what appears to be the first example of a posttranslational modification associated with turnover. Complete activation occurs in vivo when sarcosine oxidase is produced in Corynebacterium sp. P-1, where enzyme synthesis is induced by growth of the organism with sarcosine as the source of carbon and energy.(ABSTRACT TRUNCATED AT 400 WORDS) << Less

  Biochemistry 32:11132-11142(1993) [PubMed] [EuropePMC]

• Corynebacterium sarcosine oxidase: a unique enzyme having covalently-bound and noncovalently-bound flavins.

  Hayashi S., Nakamura S., Suzuki M.

  Biochem. Biophys. Res. Commun. 96:924-930(1980) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• Purification and some properties of sarcosine oxidase from Corynebacterium sp. U-96.

  Suzuki M.

  J. Biochem. 89:599-607(1981) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.