Sarcosine oxidase contains a novel covalently bound FMN.
Sarcosine oxidase from Corynebacterium sp. P-1 is a heterotetrameric protein containing three different enzymes: noncovalent FAD, noncovalent NAD+, and covalently bound flavin which is released as 8 alpha-(N3-histidyl)riboflavin upon complete hydrolysis of the protein. The following results show that the covalent flavin is not at the FAD level, as previously proposed, but it is rather as 8 alpha-(N3- histidyl)FMN coenzyme. First, no AMP is released when the protein moiety is treated with phosphodiesterase or subjected to mild acid hydrolysis. The enzyme contains a total of 5 mol of phosphate. Only one phosphate is covalently bound. The other four phosphates are noncovalent and attributed to noncovalently bound FAD and NAD+. The 31P NMR spectrum of native enzyme exhibits resonances due to a single phosphate monoester an two pyrophosphates. Only a resonance due to phosphate monoester is observed after removal of the noncovalent cofactors and proteolytic digestion of the protein moiety. The 8 alpha-(N3-histidyl)FMN found in corynebacterial sarcosine oxidase represents a novel type of covalent flavin. Studies with sarcosine oxidases from Arthrobacter sp. and Pseudomonas sp. show that these heterotetrameric enzymes also contain covalently bound FMN plus noncovalently bound FAD and NAD+, similar to corynebacterial sarcosine oxidase. In contrast, two monomeric sarcosine oxidases (from Bacillus sp. and an unidentified microorganism) were found to contain only covalently bound FAD.
Willie
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Department of Biochemistry, Medical College of Pennsylvania, Philadelphia, USA.
Edmondson
D E
DE
Jorns
M S
MS
D000249:Adenosine Monophosphate / Q000032:analysis; D002851:Chromatography, High Pressure Liquid; D003352:Corynebacterium / Q000201:enzymology* / Q000235:genetics; D005486:Flavin Mononucleotide / Q000032:analysis*; D005182:Flavin-Adenine Dinucleotide / Q000032:analysis; D006868:Hydrolysis; D009682:Magnetic Resonance Spectroscopy; D009249:NADP / Q000032:analysis; D010755:Organophosphates / Q000032:analysis; D010089:Oxidoreductases, N-Demethylating / Q000737:chemistry* / Q000187:drug effects / Q000235:genetics; D010727:Phosphoric Diester Hydrolases / Q000494:pharmacology; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry; D050879:Sarcosine Oxidase; D012697:Serine Endopeptidases / Q000494:pharmacology; D013053:Spectrophotometry
D010755:Organophosphates; D011994:Recombinant Proteins; D005182:Flavin-Adenine Dinucleotide; D000249:Adenosine Monophosphate; D009249:NADP; D005486:Flavin Mononucleotide; D010089:Oxidoreductases, N-Demethylating; D050879:Sarcosine Oxidase; D010727:Phosphoric Diester Hydrolases; D012697:Serine Endopeptidases