title: An α/β-hydrolase fold protein in the biosynthesis of thiostrepton exhibits a dual activity for endopeptidyl hydrolysis and epoxide ring opening/macrocyclization.
abstract: Thiostrepton (TSR), an archetypal bimacrocyclic thiopeptide antibiotic that arises from complex posttranslational modifications of a genetically encoded precursor peptide, possesses a quinaldic acid (QA) moiety within the side-ring system of a thiopeptide-characteristic framework. Focusing on selective engineering of the QA moiety, i.e., by fluorination or methylation, we have recently designed and biosynthesized biologically more active TSR analogs. Using these analogs as chemical probes, we uncovered an unusual indirect mechanism of TSR-type thiopeptides, which are able to act against intracellular pathogens through host autophagy induction in addition to direct targeting of bacterial ribosome. Herein, we report the accumulation of 6'-fluoro-7', 8'-epoxy-TSR, a key intermediate in the preparation of the analog 6'-fluoro-TSR. This unexpected finding led to unveiling of the TSR maturation process, which involves an unusual dual activity of TsrI, an α/β-hydrolase fold protein, for cascade C-N bond cleavage and formation during side-ring system construction. These two functions of TsrI rely on the same catalytic triad, Ser72-His200-Asp191, which first mediates endopeptidyl hydrolysis that occurs selectively between the residues Met-1 and Ile1 for removal of the leader peptide and then triggers epoxide ring opening for closure of the QA-containing side-ring system in a regio- and stereo-specific manner. The former reaction likely requires the formation of an acyl-Ser72 enzyme intermediate; in contrast, the latter is independent of Ser72. Consequently, C-6' fluorination of QA lowers the reactivity of the epoxide intermediate and, thereby, allows the dissection of the TsrI-associated enzymatic process that proceeds rapidly and typically is difficult to be realized during TSR biosynthesis.
authors.affiliation: State Key Laboratory of Bioorganic and Natural Products Chemistry, Shanghai Institute of Organic Chemistry, University of Chinese Academy of Sciences, Shanghai 200032, China.
authors.forename: Qingfei
authors.initials: Q
authors.lastname: Zheng
authors.affiliation: State Key Laboratory of Bioorganic and Natural Products Chemistry, Shanghai Institute of Organic Chemistry, University of Chinese Academy of Sciences, Shanghai 200032, China.
authors.forename: Shoufeng
authors.initials: S
authors.lastname: Wang
authors.affiliation: State Key Laboratory of Bioorganic and Natural Products Chemistry, Shanghai Institute of Organic Chemistry, University of Chinese Academy of Sciences, Shanghai 200032, China.
authors.forename: Panpan
authors.initials: P
authors.lastname: Duan
authors.affiliation: State Key Laboratory of Bioorganic and Natural Products Chemistry, Shanghai Institute of Organic Chemistry, University of Chinese Academy of Sciences, Shanghai 200032, China.
authors.forename: Rijing
authors.initials: R
authors.lastname: Liao
authors.affiliation: State Key Laboratory of Microbial Metabolism, School of Life Science & Biotechnology, Shanghai Jiao Tong University, Shanghai 200240, China.|Huzhou Center of Bio-Synthetic Innovation, Huzhou 313000, China.
authors.forename: Dandan
authors.initials: D
authors.lastname: Chen
authors.affiliation: State Key Laboratory of Bioorganic and Natural Products Chemistry, Shanghai Institute of Organic Chemistry, University of Chinese Academy of Sciences, Shanghai 200032, China; wliu@mail.sioc.ac.cn.|State Key Laboratory of Microbial Metabolism, School of Life Science & Biotechnology, Shanghai Jiao Tong University, Shanghai 200240, China.|Huzhou Center of Bio-Synthetic Innovation, Huzhou 313000, China.
authors.forename: Wen
authors.initials: W
authors.lastname: Liu
mesh_terms: D000900:Anti-Bacterial Agents / Q000096:biosynthesis / Q000737:chemistry; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D053898:Biosynthetic Pathways; D020134:Catalytic Domain; D005285:Fermentation; D006867:Hydrolases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006868:Hydrolysis; D013302:Streptomyces / Q000201:enzymology / Q000235:genetics; D013379:Substrate Specificity; D013883:Thiostrepton / Q000096:biosynthesis* / Q000737:chemistry
chemical_list: D000900:Anti-Bacterial Agents; D001426:Bacterial Proteins; D006867:Hydrolases; D013883:Thiostrepton
keywords: dual activity; endopeptidyl hydrolysis; macrocyclization; thiopeptide; α/β-hydrolase fold protein