Discovery of a third coenzyme in sarcosine oxidase.
Denaturation of recombinant sarcosine oxidase or the natural enzyme isolated from Corynebacterium sp. P-1 with guanidine hydrochloride releases noncovalently bound FAD and a second UV-absorbing component (peak 2) which comigrates with NAD+ during reversed-phase HPLC. Both FAD and peak 2 are also found in extracts prepared by incubating sarcosine oxidase at 37 degrees C for 30 min, a procedure which causes partial (approximately 50%) release of the enzyme's noncovalently bound FAD. Peak 2 in the 37 degrees C extract is heat labile and decomposes upon boiling 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 phosphodiesterase yields nicotinamide mononucleotide, AMP, and FMN, as expected for a mixture containing NAD+ and FAD. Peak 2 was converted to NADH upon reaction of the 37 degrees C extract with yeast alcohol dehydrogenase in the presence of ethanol. Guanidine hydrochloride extracts, 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 NAD+. The NAD+ is tightly bound and is not lost during enzyme purification. It is not susceptible toward hydrolysis by NADase, reduction by 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.
Department of Biochemistry, Medical College of Pennsylvania, Philadelphia 19102, USA.
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D002851:Chromatography, High Pressure Liquid; D003067:Coenzymes / Q000302:isolation & purification*; D003352:Corynebacterium / Q000201:enzymology / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D005182:Flavin-Adenine Dinucleotide / Q000302:isolation & purification; D009243:NAD / Q000302:isolation & purification / Q000378:metabolism; D010084:Oxidation-Reduction; D010089:Oxidoreductases, N-Demethylating / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011489:Protein Denaturation; D011994:Recombinant Proteins / Q000302:isolation & purification; D050879:Sarcosine Oxidase
D003067:Coenzymes; D011994:Recombinant Proteins; D009243:NAD; D005182:Flavin-Adenine Dinucleotide; D010089:Oxidoreductases, N-Demethylating; D050879:Sarcosine Oxidase