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

Author(s)

Cardoso, Tabata P. ; de Sa, Larissa A. ; Bury, Priscila dos S. ; Chavez-Pacheco, Sair M. ; Dias, Marcio V. B.

• Document type: Journal article
• Source: Protein Expression and Purification; v. 132, p. 9-18, APR 2017.
• Web of Science Citations: 0

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 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. (AU)

• FAPESP's process: 10/15971-3 - Structural characterization of enzymes from antibiotic biosynthetic pathways with biotechnological interest
• Grantee: Marcio Vinicius Bertacine Dias
• Support Opportunities: Research Grants - Young Investigators Grants
• FAPESP's process: 12/23427-7 - Structural analysis of halogenases identified in biosynthetic gene clusters of glycopeptide antibiotics.
• Grantee: Tábata Peres Cardoso
• Support Opportunities: Scholarships in Brazil - Master
• FAPESP's process: 12/16631-7 - Obtainment and crystallization of glycosyltransferases of biotechnological interest of the macrolide antibiotic biosynthesis
• Grantee: Larissa Antelo de Sá
• Support Opportunities: Scholarships in Brazil - Scientific Initiation