Cloning, expression, purification and biophysical analysis of two putative halogenases from the glycopeptide A47,934 gene cluster of Streptomyces toyocaensis

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dc.contributor.authorCardoso, Tabata P. [UNESP]
dc.contributor.authorSa, Larissa A. de
dc.contributor.authorBury, Priscila dos S.
dc.contributor.authorChavez-Pacheco, Sair M.
dc.contributor.authorDias, Marcio V. B. [UNESP]
dc.contributor.institutionCtr Nacl Pesquisa Energia & Mat
dc.contributor.institutionUniversidade Estadual Paulista (Unesp)
dc.contributor.institutionUniversidade de São Paulo (USP)
dc.date.accessioned2018-11-26T17:31:24Z
dc.date.available2018-11-26T17:31:24Z
dc.date.issued2017-04-01
dc.description.abstractGlycopeptides are an important class of antibiotics used in the treatment of several infections, including those caused by methicillin resistant Staphylococcus aureus. Glycopeptides are biosynthesized by a Non Ribosomal Peptide Synthase (NRPS) and the resulting peptide precursors are decorated by several tailoring enzymes, such as halogenases and glycosyltransferases. These enzymes are important targets of protein engineering to produce new derivatives of known antibiotics. Herein we show the production of two putative halogenases, denominated Stal and StaK, involved in the biosynthesis of the glycopeptide A47,934 in Streptomyces toyocaensis NRRL 15,009. This antibiotic together with the compound UK-68,597 are the unique glycopeptides which have two putative halogenases identified in their gene clusters and three chloride substituent atoms attached to their aglycones. Stal and StaK were successfully produced in E. coli in the soluble fraction with high purity using the wild type gene for Stal and a synthetic codon optimized gene for StaK. We have purified both enzymes by two chromatographic steps and a good yield was obtained. These putative halogenases were co-purified with the co-factor FAD, which are differently reduced by the enzyme SsuE in vitro. We have further confirmed that these putative halogenases are monomeric using a calibrated gel filtration column and through circular dichroism, we confirmed that both enzymes are folded with a predominance of alpha-helices. Molecular models for Stal and StaK were generated and together with sequence and phylogenetic analysis, we could infer some structural insights of Stal and StaK from the biosynthesis of compound A47,934. (C) 2017 Elsevier Inc. All rights reserved.en
dc.description.affiliationCtr Nacl Pesquisa Energia & Mat, Lab Nacl Biociencias, Campinas, SP, Brazil
dc.description.affiliationUniv Estadual Paulista, Inst Biociencias Letras & Ciencias Exatas, Programa Posgrad Microbiol, Sao Jose Do Rio Preto, SP, Brazil
dc.description.affiliationUniv Sao Paulo SP, Inst Ciencias Biomed, Sao Paulo, Brazil
dc.description.affiliationUnespUniv Estadual Paulista, Inst Biociencias Letras & Ciencias Exatas, Programa Posgrad Microbiol, Sao Jose Do Rio Preto, SP, Brazil
dc.description.sponsorshipFundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)
dc.description.sponsorshipCoordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)
dc.description.sponsorshipIdFAPESP: 2010/15971-3
dc.description.sponsorshipId: 2012/23427-7
dc.description.sponsorshipId: 2012/16631-7
dc.format.extent9-18
dc.identifierhttp://dx.doi.org/10.1016/j.pep.2017.01.001
dc.identifier.citationProtein Expression And Purification. San Diego: Academic Press Inc Elsevier Science, v. 132, p. 9-18, 2017.
dc.identifier.doi10.1016/j.pep.2017.01.001
dc.identifier.fileWOS000401298600002.pdf
dc.identifier.issn1046-5928
dc.identifier.urihttp://hdl.handle.net/11449/162789
dc.identifier.wosWOS:000401298600002
dc.language.isoeng
dc.publisherElsevier B.V.
dc.relation.ispartofProtein Expression And Purification
dc.relation.ispartofsjr0,648
dc.rights.accessRightsAcesso aberto
dc.sourceWeb of Science
dc.subjectHalogenase
dc.subjectGlycopeptide biosynthesis
dc.subjectStreptomyces toyocaensis
dc.titleCloning, expression, purification and biophysical analysis of two putative halogenases from the glycopeptide A47,934 gene cluster of Streptomyces toyocaensisen
dc.typeArtigo
dcterms.licensehttp://www.elsevier.com/about/open-access/open-access-policies/article-posting-policy
dcterms.rightsHolderElsevier B.V.

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