SARCOSINE OXIDASE CONTAINS A NOVEL COVALENTLY BOUND FMN

Sarcosine oxidase from Corynebacterium sp. P-1 is a heterotetrameric p rotein containing three different coenzymes, noncovalent FAD, noncoval ent NAD(+), and a covalently bound flavin which is released as 8 alpha -(N-3-histidyl)riboflavin upon complete hydrolysis of the protein, The following results show that the covalent flavin is not at the FAD lev el, as previously proposed, but is rather an 8 alpha-(N-3-histidyl)FMN coenzyme. First, no AMP is released when the protein moiety is treate d with phosphodiesterase or subjected to mild acid hydrolysis. The enz yme contains a total of 5 mol of phosphate, Only one phosphate is cova lently bound. The other four phosphates are noncovalent and attributed to noncovalently bound FAD and NAD(+), The P-31 NMR spectrum of nativ e enzyme exhibits resonances due to a single phosphate monoester and t wo pyrophosphates, Only a resonance due to phosphate monoester is obse rved after removal of the noncovalent cofactors and proteolytic digest ion of the protein moiety, The 8 alpha-(N-3-histidyl)FMN found in cory nebacterial sarcosine oxidase represents a novel type of covalent flav in, Studies with sarcosine oxidases from Arthrobacter sp. and Pseudomo nas sp, show that these heterotetrameric enzymes also contain covalent ly bound FMN plus noncovalently bound FAD and NAD(+), similar to coryn ebacterial sarcosine oxidase. In contrast, two monomeric sarcosine oxi dases (from Bacillus sp, and an unidentified microorganism) were found to contain only covalently bound FAD.