Structure and Substrate Recognition of the Bottromycin Maturation Enzyme BotP

Abstract

Abstract:The bottromycins are a family of highly modified peptide natural products displaying potent antimicrobial activity against Gram-positive bacteria including methicillin-resistant Staphyloccoccus aureus. Bottromycins have recently been shown to be ribosomally synthesized and post-translationally modified peptides (RiPPs). Uniquely amongst RiPPs the precursor peptide BotA contains a C-terminal follower, rather than the canonical Nterminal leader sequence. We report the structural and biochemical characterization of BotP, a leucyl-aminopeptidase like enzyme from the bottromycin pathway. We demonstrate that BotP is responsible for the removal of the N-terminal methionine from the precursor peptide. The crystal structures of apo BotP and of BotP in complex with Mn 2+ allowed us to model a BotP/substrate complex and to rationalize substrate recognition. Our data represent the first step towards targeted compound modification to unlock the full antibiotic potential of bottromycin.

Thiazoline-Specific Amidohydrolase PurAH Is the Gatekeeper of Bottromycin Biosynthesis

“…In vitro work has confirmed the assigned functions for BotP [13][14] , BotC [8][9] and BotCD [8][9] . It was demonstrated that the BotCD homologs BmbE and PurCD alone are sufficient for macroamidine formation in vitro [8][9] .…”

New developments in RiPP discovery, enzymology and engineering

“…[138][139][140][141] Gene deletion studies conducted on various bottromycinproducing strains assigned the role of Asp methylester-ication to the O-methyltransferase and b-methylation of Phe, Val, and Pro to the three class B radical SAM methyltransferases found in the BGC. 138,141 Biochemical studies showed that the leucyl-aminopeptidase BmbK removes the N-terminal Met 142,143 and conrmed that BmbB catalyzes O-methylation of Asp. 143 An untargeted metabolomics study carried out on mutants of the BGC in Streptomyces scabies proposed an order for the post-translational modications in bottromycin biosynthesis as detailed in Fig.…”

Macroamidine Formation in Bottromycins Is Catalyzed by a Divergent YcaO Enzyme

“…When BotA was incubated with IpoC and ATP/Mg 2+ , we observed a loss of 18 Da consistent with cyclodehydration (Figure S4a), and the conversion of Cys9 to thiazoline was confirmed by tandem MS (MS 2+ ) (Figure S4b and Table S2). We treated BotA with BotP (BotA P ) to make the glycine amino group available as a nucleophile for macrocyclization and repeated the experiments . The reaction with IpoC also resulted in thiazoline formation at Cys9 (Figures and S5, Table S4).…”