The mildiomycin biosynthesis: Initial steps for sequential generation of 5-hydroxymethylcytidine 5′-monophosphate and 5-hydroxymethylcytosine in Streptoverticillium rimofaciens ZJU5119

Li Li, Zhinan Xu, Xiaoying Xu, Jun Wu, Yun Zhang, Xinyi He, Mark Zabriskie, Zixin Deng

Research output: Contribution to journalArticle

40 Citations (Scopus)

Abstract

Mildiomycin (MIL) is a peptidyl nucleoside antibiotic with strong activity against powdery mildew disease of plants. We have cloned the MIL biosynthetic gene cluster in Streptoverticillum rimofaciens ZJU5119 and shown that this organism also produces the related antifungal compound, deshydroxymethyl mildiomycin (dHM-MIL). A cosmid genomic library was screened for a putative nucleotide hydrolase gene that is related to blsM from the blasticidin S cluster. Six cosmids were identified that contained a 3.5 kb DNA fragment that harbors a homologue of blsM. The sequence of the fragment revealed two open-reading frames that are likely to function in MIL formation: milA is a CMP hydroxymethylase gene and milB is the homologue of the CMP hydrolase gene blsM. Insertional disruption of milA abolished the production of MIL but not dHM-MIL, whereas a milB knockout strain did not produce either of the peptidyl nucleosides. Recombinant MilA was produced in E. coli and shown to specifically introduce a C-5 hydroxymethyl group on CMP, but it did not accept cytosine or dCMP as a substrate. MilB was also expressed and purified from E. coli and shown to efficiently hydrolyze both hydroxymethyl-CMP (HMCMP) and could accept CMP as an alternative substrate. The ratio of free HMC and cytosine released by MilB was ca. 9:1 in in vitro assays, and is consistent with the higher levels of MIL compared to dHM-MIL that are produced by Streptoverticillum rimofaciens.

Original languageEnglish (US)
Pages (from-to)1286-1294
Number of pages9
JournalChemBioChem
Volume9
Issue number8
DOIs
StatePublished - May 23 2008
Externally publishedYes

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Biosynthesis
Cytidine Monophosphate
Genes
Cosmids
Cytosine
Hydrolases
Nucleosides
Escherichia coli
Plant Diseases
Genomic Library
5-hydroxymethylcytidine 5'-monophosphate
5-hydroxymethylcytosine
mildiomycin
Substrates
Multigene Family
Ports and harbors
Open Reading Frames
Assays
Nucleotides
Anti-Bacterial Agents

Keywords

  • Antifungal agents
  • Biosynthesis
  • Nucleotide hydrolase
  • Peptidyl nucleoside
  • Pyrimidine hydroxymethylase

ASJC Scopus subject areas

  • Biochemistry
  • Organic Chemistry
  • Molecular Medicine
  • Molecular Biology
  • Medicine(all)

Cite this

The mildiomycin biosynthesis : Initial steps for sequential generation of 5-hydroxymethylcytidine 5′-monophosphate and 5-hydroxymethylcytosine in Streptoverticillium rimofaciens ZJU5119. / Li, Li; Xu, Zhinan; Xu, Xiaoying; Wu, Jun; Zhang, Yun; He, Xinyi; Zabriskie, Mark; Deng, Zixin.

In: ChemBioChem, Vol. 9, No. 8, 23.05.2008, p. 1286-1294.

Research output: Contribution to journalArticle

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title = "The mildiomycin biosynthesis: Initial steps for sequential generation of 5-hydroxymethylcytidine 5′-monophosphate and 5-hydroxymethylcytosine in Streptoverticillium rimofaciens ZJU5119",
abstract = "Mildiomycin (MIL) is a peptidyl nucleoside antibiotic with strong activity against powdery mildew disease of plants. We have cloned the MIL biosynthetic gene cluster in Streptoverticillum rimofaciens ZJU5119 and shown that this organism also produces the related antifungal compound, deshydroxymethyl mildiomycin (dHM-MIL). A cosmid genomic library was screened for a putative nucleotide hydrolase gene that is related to blsM from the blasticidin S cluster. Six cosmids were identified that contained a 3.5 kb DNA fragment that harbors a homologue of blsM. The sequence of the fragment revealed two open-reading frames that are likely to function in MIL formation: milA is a CMP hydroxymethylase gene and milB is the homologue of the CMP hydrolase gene blsM. Insertional disruption of milA abolished the production of MIL but not dHM-MIL, whereas a milB knockout strain did not produce either of the peptidyl nucleosides. Recombinant MilA was produced in E. coli and shown to specifically introduce a C-5 hydroxymethyl group on CMP, but it did not accept cytosine or dCMP as a substrate. MilB was also expressed and purified from E. coli and shown to efficiently hydrolyze both hydroxymethyl-CMP (HMCMP) and could accept CMP as an alternative substrate. The ratio of free HMC and cytosine released by MilB was ca. 9:1 in in vitro assays, and is consistent with the higher levels of MIL compared to dHM-MIL that are produced by Streptoverticillum rimofaciens.",
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T2 - Initial steps for sequential generation of 5-hydroxymethylcytidine 5′-monophosphate and 5-hydroxymethylcytosine in Streptoverticillium rimofaciens ZJU5119

AU - Li, Li

AU - Xu, Zhinan

AU - Xu, Xiaoying

AU - Wu, Jun

AU - Zhang, Yun

AU - He, Xinyi

AU - Zabriskie, Mark

AU - Deng, Zixin

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N2 - Mildiomycin (MIL) is a peptidyl nucleoside antibiotic with strong activity against powdery mildew disease of plants. We have cloned the MIL biosynthetic gene cluster in Streptoverticillum rimofaciens ZJU5119 and shown that this organism also produces the related antifungal compound, deshydroxymethyl mildiomycin (dHM-MIL). A cosmid genomic library was screened for a putative nucleotide hydrolase gene that is related to blsM from the blasticidin S cluster. Six cosmids were identified that contained a 3.5 kb DNA fragment that harbors a homologue of blsM. The sequence of the fragment revealed two open-reading frames that are likely to function in MIL formation: milA is a CMP hydroxymethylase gene and milB is the homologue of the CMP hydrolase gene blsM. Insertional disruption of milA abolished the production of MIL but not dHM-MIL, whereas a milB knockout strain did not produce either of the peptidyl nucleosides. Recombinant MilA was produced in E. coli and shown to specifically introduce a C-5 hydroxymethyl group on CMP, but it did not accept cytosine or dCMP as a substrate. MilB was also expressed and purified from E. coli and shown to efficiently hydrolyze both hydroxymethyl-CMP (HMCMP) and could accept CMP as an alternative substrate. The ratio of free HMC and cytosine released by MilB was ca. 9:1 in in vitro assays, and is consistent with the higher levels of MIL compared to dHM-MIL that are produced by Streptoverticillum rimofaciens.

AB - Mildiomycin (MIL) is a peptidyl nucleoside antibiotic with strong activity against powdery mildew disease of plants. We have cloned the MIL biosynthetic gene cluster in Streptoverticillum rimofaciens ZJU5119 and shown that this organism also produces the related antifungal compound, deshydroxymethyl mildiomycin (dHM-MIL). A cosmid genomic library was screened for a putative nucleotide hydrolase gene that is related to blsM from the blasticidin S cluster. Six cosmids were identified that contained a 3.5 kb DNA fragment that harbors a homologue of blsM. The sequence of the fragment revealed two open-reading frames that are likely to function in MIL formation: milA is a CMP hydroxymethylase gene and milB is the homologue of the CMP hydrolase gene blsM. Insertional disruption of milA abolished the production of MIL but not dHM-MIL, whereas a milB knockout strain did not produce either of the peptidyl nucleosides. Recombinant MilA was produced in E. coli and shown to specifically introduce a C-5 hydroxymethyl group on CMP, but it did not accept cytosine or dCMP as a substrate. MilB was also expressed and purified from E. coli and shown to efficiently hydrolyze both hydroxymethyl-CMP (HMCMP) and could accept CMP as an alternative substrate. The ratio of free HMC and cytosine released by MilB was ca. 9:1 in in vitro assays, and is consistent with the higher levels of MIL compared to dHM-MIL that are produced by Streptoverticillum rimofaciens.

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