Gene sequence and properties of an s-triazine ring-cleavage enzyme from Pseudomonas sp strain NRRLB-12227

Pesticides based on the s-triazine ring structure are widely used in cultiv ation of food crops. Cleavage of the s-triazine ring is an important step i n the mineralization of s-triazine compounds and hence in their complete re moval from the environment. Cyanuric acid amidohydrolase cleaves cyanuric a cid (2,4,6-trihydroxy-s-triazine), which yields carbon dioxide and biuret; the biuret is subject to further metabolism, which yields CO2 and ammonia. The trzD gene encoding cyanuric acid amidohydrolase was cloned into pMMB277 from Pseudomonas sp. strain NRRLB-12227, a strain that is capable of utili zing s-triazines as nitrogen sources. Hydrolysis of cyanuric acid was detec ted in crude extracts of Escherichia coli containing the cloned gene by mon itoring the disappearance of cyanuric acid and the appearance of biuret by high-performance liquid chromatography (HPLC). DEAE and hydrophobic interac tion HPLC were used to purify cyanuric acid amidohydrolase to homogeneity a nd a spectrophotometric assay for the purified enzyme was developed. The pu rified enzyme had an apparent K-m of 0.05 mM for cyanuric acid at pH 8.0. T he enzyme did not cleave any other s-triazine or hydropyrimidine compound, although barbituric acid (2,4,6-trihydroxypyrimidine) was found to be a str ong competitive inhibitor. Neither the nucleotide sequence of trzD nor the amino acid sequence of the gene product exhibited a significant level of si milarity to any known gene or protein.