Characterization of BIsM, a nucleotide hydrolase involved in cytosine production for the biosynthesis of blasticidin S

Laura L. Grochowski, Mark Zabriskie

Research output: Contribution to journalArticle

12 Citations (Scopus)

Abstract

Biosynthesis of the antifungal agent blasticidin S in Streptomyces griseochromogenes requires the formation of free cytosine. The bIsM 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 BIsM 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. BIsM was also observed to have low-level cytidine deaminase activity, converting cytidine and deoxycytidine to uridine and deoxyuridine, respectively. Point mutations were introduced in bIsM at putative catalytic residues to generate three mutant enzymes, BIsM Ser98Asp, Glu104Ala, and Glu104Asp. All three mutants lost CMP hydrolysis activity, but the Ser98Asp mutant showed a modest increase in cytidine deaminase activity.

Original languageEnglish (US)
Pages (from-to)957-964
Number of pages8
JournalChemBioChem
Volume7
Issue number6
DOIs
StatePublished - Jun 2006
Externally publishedYes

Fingerprint

Cytidine
Cytosine
Biosynthesis
Hydrolases
Cytidine Deaminase
Cytidine Monophosphate
Nucleotides
Genes
Cytidine Diphosphate
Cytidine Triphosphate
Amino Acid Sequence Homology
Deoxyuridine
Deoxycytidine
Antifungal Agents
Uridine
Streptomyces
Substrates
Enzymes
Multigene Family
Transferases

Keywords

  • Antifungal agents
  • Biosynthesis
  • Cytosine
  • Hydrolases
  • Nucleosides

ASJC Scopus subject areas

  • Drug Discovery
  • Organic Chemistry
  • Biochemistry, Genetics and Molecular Biology(all)

Cite this

Characterization of BIsM, a nucleotide hydrolase involved in cytosine production for the biosynthesis of blasticidin S. / Grochowski, Laura L.; Zabriskie, Mark.

In: ChemBioChem, Vol. 7, No. 6, 06.2006, p. 957-964.

Research output: Contribution to journalArticle

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N2 - Biosynthesis of the antifungal agent blasticidin S in Streptomyces griseochromogenes requires the formation of free cytosine. The bIsM 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 BIsM 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. BIsM was also observed to have low-level cytidine deaminase activity, converting cytidine and deoxycytidine to uridine and deoxyuridine, respectively. Point mutations were introduced in bIsM at putative catalytic residues to generate three mutant enzymes, BIsM Ser98Asp, Glu104Ala, and Glu104Asp. All three mutants lost CMP hydrolysis activity, but the Ser98Asp mutant showed a modest increase in cytidine deaminase activity.

AB - Biosynthesis of the antifungal agent blasticidin S in Streptomyces griseochromogenes requires the formation of free cytosine. The bIsM 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 BIsM 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. BIsM was also observed to have low-level cytidine deaminase activity, converting cytidine and deoxycytidine to uridine and deoxyuridine, respectively. Point mutations were introduced in bIsM at putative catalytic residues to generate three mutant enzymes, BIsM Ser98Asp, Glu104Ala, and Glu104Asp. All three mutants lost CMP hydrolysis activity, but the Ser98Asp mutant showed a modest increase in cytidine deaminase activity.

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