Genes encoding ribonucleoside hydrolase 1 and 2 from Corynebacterium ammoniagenes

Abstract

Two kinds of nucleoside hydrolases (NHs) encoded byrih1andrih2were cloned fromCorynebacterium ammoniagenesusingdeoD- andgsk-defectiveEscherichia coli. Sequence analysis revealed that NH 1 was a protein of 337 aa with a deduced molecular mass of 35 892 Da, whereas NH 2 consisted of 308 aa with a calculated molecular mass of 32 310 Da. Experiments with crude extracts of IPTG-inducedE. coliCGSC 6885(pTNU23) and 6885(pTNI12) indicated that the Rih1 enzyme could catalyse the hydrolysis of uridine and cytidine and showed pyrimidine-specific ribonucleoside hydrolase activity. Rih2 was able to hydrolyse both purine and pyrimidine ribonucleosides with the following order of activity – inosine>adenosine>uridine>guanosine>xanthosine>cytidine – and was classified in the non-specific NHs family.rih1andrih2deletion mutants displayed a decrease in cell growth on minimal medium supplemented with pyrimidine and purine/pyrimidine nucleosides, respectively, compared with the wild-type strain. Growth of each mutant was substantially complemented by introducingrih1andrih2, respectively. Furthermore, disruption of bothrih1andrih2led to the inability of the mutant to utilize purine and pyrimidine nucleosides as sole carbon source on minimal medium. These results indicated thatrih1andrih2play major roles in the salvage pathways of nucleosides in this micro-organism.