Search for Microbial Enzymes Useful for Creatinine Assay

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A novel metabolic pathway for the degradation of creatinine with N-methylhydantoin, N-carbamoylsarcosine and sarcosine as successive intermediates was found to operate in Pseudomonas putida 77 and many other microorganisms. Enzymes involved in this pathway were purified from cells of P. Putida and characterized. The first step, deimination of creatinine, is catalyzed by cytosine deaminase/creatinine deiminase. The following two steps, ring-openinig of N-methylhydantoin and decarbamoylation of N-carbamoylsarcosine, are catalyzed by new enzymes, N-methylgydantoin amidohydrolase and N-carbamoylsarcosine amidohydrolase, respectively. The former enzyme requires ATP, Mg^<2+>, and for the hydrolysis and the reaction proceeds as follows : N-methylhydantoin ---> N-carbamoylsarcosine + ADP + Pi. The latter catalyzes the following reaction ; N-carbamoylsarcosine + H_20 ---> sarcosine + NH. Sarcosine dehydrogenase was found to be the responsible enzyme for the oxidation of sarcosine to glycine in P. Putida 77, but sarcosine oxidase was also found to be involved in this oxidation in several microorganisms. These enzymes were found to be useful for determination of creatinine.