DISCOVERY OF A 3RD COENZYME IN SARCOSINE OXIDASE

Denaturation of recombinant sarcosine oxidase or the natural enzyme is olated from Corynebacterium sp. P-1 with guanidine hydrochloride relea ses noncovalently bound FAD and a second UV-absorbing component (peak 2) which comigrates with NAD(+) during reversed-phase HPLC. Both FAD a nd peak 2 are also found in extracts prepared by incubating sarcosine oxidase at 37 degrees C for 30 min, a procedure which causes partial ( similar to 50%) release of the enzyme's noncovalently bound FAD. Peak 2 in the 37 degrees C extract is heat labile and decomposes upon boili ng for 5 min at pH 8.0. A similar instability was observed with NAD(+) . Reaction of the 37 degrees C extract from sarcosine oxidase with pho sphodiesterase yields nicotinamide mononucleotide, AMP, and FMN, as ex pected for a mixture containing NAD(+) and FAD. Peak 2 was converted t o NADH upon reaction of the 37 degrees C extract with yeast alcohol de hydrogenase in the presence of ethanol. Guanidine hydrochloride extrac ts, prepared from recombinant or natural enzyme, contain 1 mol of NAD( +)/mol of FAD. Since sarcosine oxidase contains 1 mol of noncovalently bound FAD, the results show that the enzyme also contains 1 mol of NA D(+). The NAD(+) is tightly bound and is not lost during enzyme purifi cation. It is not susceptible toward hydrolysis by NADase, reduction b y alcohol dehydrogenase, or nucleophilic attack by cyanide. Unlike the flavins in sarcosine oxidase, NAD(+) is not reduced by sarcosine and is not in redox equilibrium with the flavins.