Genes encoding ribonucleoside hydrolase 1 and 2 from Corynebacterium ammoniagenes.
Two kinds of nucleoside hydrolases (NHs) encoded by rih1 and rih2 were cloned from Corynebacterium ammoniagenes using deoD- and gsk-defective Escherichia 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-induced E. coli CGSC 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. rih1 and rih2 deletion 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 introducing rih1 and rih2, respectively. Furthermore, disruption of both rih1 and rih2 led to the inability of the mutant to utilize purine and pyrimidine nucleosides as sole carbon source on minimal medium. These results indicated that rih1 and rih2 play major roles in the salvage pathways of nucleosides in this micro-organism.
Kim
Hyun-Soo
HS
R&D Center for Bioproducts, CJ Corp., Seoul 157-724, Korea.
Lee
Jin-Ho
JH
R&D Center for Bioproducts, CJ Corp., Seoul 157-724, Korea.
Lee
Won-Sik
WS
R&D Center for Bioproducts, CJ Corp., Seoul 157-724, Korea.
Bang
Won-Gi
WG
Department of Biotechnology and Genetic Engineering, College of Life and Environmental Sciences, Korea University, Anam-dong, Seoul 136-701, Korea.
D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D003352:Corynebacterium / Q000201:enzymology / Q000235:genetics* / Q000502:physiology; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D017353:Gene Deletion; D005816:Genetic Complementation Test; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009699:N-Glycosyl Hydrolases / Q000737:chemistry / Q000235:genetics*; D016366:Open Reading Frames; D011687:Purines / Q000378:metabolism; D011743:Pyrimidines / Q000378:metabolism; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D013379:Substrate Specificity
D004269:DNA, Bacterial; D011687:Purines; D011743:Pyrimidines; D009699:N-Glycosyl Hydrolases