Characterization of BlsM, a Nucleotide Hydrolase Involved in Cytosine Production for the Biosynthesis of Blasticidin S

Abstract

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.

Structural Characterization of the Mammalian Deoxynucleotide N-Hydrolase Rcl and Its Stabilizing Interactions with Two Inhibitors

“…Another member of this novel nucleotide monophosphate hydrolase subfamily is BlsM, which was previously shown to hydrolyze dCMP as well as CMP. 24 Sequence comparisons highlighted Rcl and BlsM as atypical members of the N-(deoxy)ribosyltransferase-like superfamily. 8 Such a divergent evolution is reminiscent to that of the nucleoside phosphorylase family composed of purine nucleoside phosphorylase (PNP), 5′-deoxy-5′-methylthioadenosine phosphorylase, and AMP nucleosidase (AMN).…”

The c-Myc Target Gene Rcl (C6orf108) Encodes a Novel Enzyme, Deoxynucleoside 5′-monophosphate N-Glycosidase

“…The biochemical experiments performed with either pure native RCL or with a His-tagged RCL demonstrate that it codes for a deoxynucleoside 5Ј-monophosphate glycosidase, a previously undescribed activity. Indeed, N-glycosylhydrolases with a pyrimidine preference have been described in Neisseria meningitidis (20), Streptomyces virginiae (21,22), and S. griseochromogenes (23,24). On the other hand, ribosylhydrolases specific for inosine or adenosine 5Ј-monophosphate were also reported (25,26).…”

“…Only Zn 2ϩ inhibited the reaction, whereas the other metals had no effect. Initial velocity experiments were carried out at variable concentrations of the six deoxynucleoside 5Ј-monophosphate: dAMP, dCMP, dGMP, dIMP, dTMP, and dUMP ( (24). To better understand the catalytic mechanism of RCL, several compounds were tested as the substrate or inhibitor of the enzyme activity.…”

Characterization of the Amicetin Biosynthesis Gene Cluster from Streptomyces vinaceusdrappus NRRL 2363 Implicates Two Alternative Strategies for Amide Bond Formation

“…4B). AmiI shows sequence similarity to various nucleoside 2-deoxyribosyltransferases that catalyze the cleavage of the glycosidic bond of 2-deoxyribonucleosides, among which BlsM (34% identity) from the blasticidin S pathway and MilB (27% identity) from the mildiomycin pathway have been functionally characterized (28,45). The amiI gene was inactivated to probe its functional role in amicetin biosynthesis, resulting in the ⌬amiI mutant AM1006.…”