Title
Cloning, expression, purification and biophysical analysis of two putative halogenases from the glycopeptide A47,934 gene cluster of Streptomyces toyocaensis
Author
Tabata P. Cardoso, Larissa A. de Sá, Priscila dos S. Bury, Sair M.C. Pacheco, Marcio V.B. Dias
Year
2017
Journal
Protein Expression and Purification
Abstract
Glycopeptides are an important class of antibiotics used in the treatment of several infections, including those ones 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 as 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 substituents 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 have been 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 of circular dicroism, 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.
Instrument
J-810
Keywords
Circular dichroism, Secondary structure, Thermal stability, Thermodynamics, Biochemistry