Characterization of recombinant monomeric sarcosine oxidase from Bacillus sp. B-0618

Sarcosine oxidases are flavoprotein oxidases which demethylate sarcosine, forming glycine and formaldehyde. Under aerobic conditions, hydrogen peroxide is generated as the enzyme flavin passes reducing equivalents to molecular oxygen. There are two major classes of bacterial sarcosine oxidases: heterotetrameric (TSOX, [alpha] = 96-103 kDa, [beta] = 42-45 kDa, [gamma] = 20-23 kDa, [delta] = 6-14 kDa) and monomeric (MSOX, 42-45 kDa). MSOX shares sequence homology with the [beta]-subunit of TSOX. A folate-usage study revealed that during catalysis, TSOX is able to transfer the methyl group of sarcosine to [6S]-tetrahydrofolate, forming 5,10-methylene-tetrahydrofolate instead of formaldehyde. MSOX is unable to use tetrahydrofolate as a one-carbon acceptor. Recombinant Bacillus sp. B-0618 MSOX was overexpressed in E. coli DH1 containing the MSOX expression vector pMAW. The enzyme was purified by ammonium sulfate fractionation and ion-exchange chromatography. The apparent Km and k cat of MSOX was determined in air-saturated solution using sarcosine, N-ethylglycine, and L-proline. The absorbance spectrum of MSOX was perturbed by several sarcosine-like and proline-like substrate analogs. Based on these perturbations, the affinity of MSOX for these compounds was measured. Chemical analysis and sequencing of the MSOX flavin peptide determined the FAD cofactor of MSOX is covalently bound to the protein through an 8[alpha]-(S-cysteinyl) linkage at Cys315. Steady-state kinetic studies, using sarcosine or N-methyl-L-alanine as substrate at various oxygen concentrations, showed that MSOX functions via a ternary complex mechanism. MSOX forms a thermodynamically stable red anionic flavin radical upon anaerobic reduction with a sub-stoichiometric concentration of sarcosine. The red radical was also seen under anaerobic conditions by EDTA light reduction, and upon incubation with dithiothreitol or thioglycolate. A slow-forming enzyme sulfite complex was observed at pH 7.0. A selenomethionine-substituted MSOX derivative was successfully created by expression of the enzyme in the pMAW-containing methionine auxotrophic host, E. coli DL41, cultured in the presence of L-selenomethionine. Purified selenomethionine-substituted MSOX was used in crystallographic studies to determine the atomic structure of MSOX. Based on primary amino acid sequence, MSOX is a member of an emerging family of flavoenzymes which use similar substrates and contain covalently bound flavin.