title: Cloning, expression, purification and biophysical analysis of two putative halogenases from the glycopeptide A47,934 gene cluster of Streptomyces toyocaensis.
abstract: Glycopeptides 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 StaI 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. StaI and StaK were successfully produced in E. coli in the soluble fraction with high purity using the wild type gene for StaI 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 α-helices. Molecular models for StaI and StaK were generated and together with sequence and phylogenetic analysis, we could infer some structural insights of StaI and StaK from the biosynthesis of compound A47,934.
authors.affiliation: Laboratório Nacional de Biociências, Centro Nacional de Pesquisa em Energia e Materais, Campinas, SP, Brazil; Programa de Pós-Graduação em Microbiologia, Instituto de Biociências, Letras e Ciências Exatas, Universidade Estadual Paulista, São José do Rio Preto, SP, Brazil.
authors.forename: Tabata P
authors.initials: TP
authors.lastname: Cardoso
authors.affiliation: Instituto de Ciências Biomédicas, Universidade de São Paulo-SP, São Paulo, Brazil.
authors.forename: Larissa A
authors.initials: LA
authors.lastname: de Sá
authors.affiliation: Instituto de Ciências Biomédicas, Universidade de São Paulo-SP, São Paulo, Brazil.
authors.forename: Priscila Dos S
authors.initials: PDS
authors.lastname: Bury
authors.affiliation: Instituto de Ciências Biomédicas, Universidade de São Paulo-SP, São Paulo, Brazil.
authors.forename: Sair M
authors.initials: SM
authors.lastname: Chavez-Pacheco
authors.affiliation: Laboratório Nacional de Biociências, Centro Nacional de Pesquisa em Energia e Materais, Campinas, SP, Brazil; Programa de Pós-Graduação em Microbiologia, Instituto de Biociências, Letras e Ciências Exatas, Universidade Estadual Paulista, São José do Rio Preto, SP, Brazil; Instituto de Ciências Biomédicas, Universidade de São Paulo-SP, São Paulo, Brazil. Electronic address: mvbdias@usp.br.
authors.forename: Marcio V B
authors.initials: MVB
authors.lastname: Dias
mesh_terms: D001426:Bacterial Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D003001:Cloning, Molecular; D015870:Gene Expression; D006023:Glycoproteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D005810:Multigene Family; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D013302:Streptomyces / Q000201:enzymology / Q000235:genetics*
chemical_list: D001426:Bacterial Proteins; D006023:Glycoproteins; D011994:Recombinant Proteins
keywords: Glycopeptide biosynthesis; Halogenase; Streptomyces toyocaensis