Characterization of BlsM, a nucleotide hydrolase involved in cytosine production for the biosynthesis of blasticidin S.
Biosynthesis of the antifungal agent blasticidin S in Streptomyces griseochromogenes requires the formation of free cytosine. The blsM gene in the blasticidin S gene cluster is predicted to encode a protein that has sequence homology with several nucleoside transferases. In vitro analysis of recombinant BlsM revealed that the enzyme functions as a nucleotide hydrolase and catalyzes the formation of free cytosine by using cytidine 5'-monophosphate (CMP) as the preferred substrate. Cytosine production was significantly lower with CDP, CTP, and dCMP as alternate substrates. BlsM was also observed to have low-level cytidine deaminase activity, converting cytidine and deoxycytidine to uridine and deoxyuridine, respectively. Point mutations were introduced in blsM at putative catalytic residues to generate three mutant enzymes, BlsM Ser98Asp, Glu104Ala, and Glu104Asp. All three mutants lost CMP hydrolysis activity, but the Ser98Asp mutant showed a modest increase in cytidine deaminase activity.
Grochowski
Laura L
LL
Department of Pharmaceutical Sciences, Oregon State University, Corvallis, OR 97331-3507, USA.
Zabriskie
T Mark
TM
D000595:Amino Acid Sequence; D000935:Antifungal Agents / Q000096:biosynthesis / Q000737:chemistry; D002240:Carbohydrate Sequence; D003596:Cytosine / Q000737:chemistry* / Q000378:metabolism; D006867:Hydrolases / Q000737:chemistry* / Q000235:genetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009705:Nucleosides / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D009711:Nucleotides / Q000737:chemistry* / Q000235:genetics; D016415:Sequence Alignment; D013302:Streptomyces / Q000201:enzymology* / Q000235:genetics; D013379:Substrate Specificity
D000935:Antifungal Agents; D009705:Nucleosides; D009711:Nucleotides; C513098:blasticidin M protein, Streptomyces; C004500:blasticidin S; D003596:Cytosine; D006867:Hydrolases