The Diiron Monooxygenase CmlA from Chloramphenicol Biosynthesis Allows Reconstitution of β-Hydroxylation during Glycopeptide Antibiotic Biosynthesis

Abstract

β-Hydroxylation plays an important role in the nonribosomal peptide biosynthesis of many important natural products, including bleomycin, chloramphenicol, and the glycopeptide antibiotics (GPAs). Various oxidative enzymes have been implicated in such a process, with the mechanism of incorporation varying from installation of hydroxyl groups in amino acid precursors prior to adenylation to direct amino acid oxidation during peptide assembly. In this work, we demonstrate the in vitro utility and scope of the unusual nonheme diiron monooxygenase CmlA from chloramphenicol biosynthesis for the β-hydroxylation of a diverse range of carrier protein bound substrates by adapting this enzyme as a non-native trans -acting enzyme within NRPS-mediated GPA biosynthesis. The results from our study show that CmlA has a broad substrate specificity for modified phenylalanine/tyrosine residues as substrates and can be used in a practical strategy to functionally cross complement compatible NRPS biosynthesis pathways in vitro .

Exploring modular reengineering strategies to redesign the teicoplanin non-ribosomal peptide synthetase

“…Here, we tested two new approaches: (1) using C/A interface reengineering between distinct modules and (2) the relocation of an E-domain to ensure communication with the downstream module. The success of these experiments, taken together with the results of our previous M6 PCP exchange experiments perform in the M6 A-PCP construct, 26 indicates that both C/A and A/PCP regions are exible in terms domain exchange. The critical factor in these bio-combinatorial experiments is the selectivity of the acceptor site of the upstream C-domain, which in teicoplanin M6 is high due to the need for this domain to gate aminoacyl-PCP modications by trans-acting enzymes.…”

“…1). 6,7,14,[25][26][27][28] Now, we explore the ability to generate alternate NRPS assembly lines from teicoplanin biosynthesis using a combination of approaches including module hybridisation, re-purposing extension modules as initiation modules and redesigning modules to control intermodule communication through the use of specic domain interaction interfaces. In this way, we present a set of instructions to tackle the reconstitution and reengineering of complex NRPS assembly lines in vitro.…”

In Vitro Reconstitution Reveals a Central Role for the N‐Oxygenase PvfB in (Dihydro)pyrazine‐N‐oxide and Valdiazen Biosynthesis

“…Communications low sequence identity with PvfC-T and PvfC Pf0-1 -T (Supporting Information, Figure S29 and Table S2), particularly in regions proposed to be involved in interactions with tailoring enzymes. [14] Structural and binding studies are underway to characterize the interactions between PvfB and PvfC Tdomains.…”

In Vitro Reconstitution Reveals a Central Role for the N‐Oxygenase PvfB in (Dihydro)pyrazine‐N‐oxide and Valdiazen Biosynthesis

“…Zuschriften low sequence identity with PvfC-T and PvfC Pf0-1 -T (Supporting Information, Figure S29 and Table S2), particularly in regions proposed to be involved in interactions with tailoring enzymes. [14] Structural and binding studies are underway to characterize the interactions between PvfB and PvfC Tdomains.…”

“…P450 sky in skyllamycin biosynthesis b-hydroxylates three different amino acids, each tethered to a different, native T-domain, [13] while CmlA, a diiron b-hydroxylase in chloramphenicol biosynthesis, can modify tyrosine derivatives tethered to a non-native Tdomain in teicoplanin biosynthesis. [14] Thus, we examined two alternate T-domains as substrates for PvfB. We used the Tdomain of PvfC from P. fluorescens Pf0-1 (PvfC Pf0-1 -T) and the T-domain of PA4078, an NRPS that comes from a pvfunrelated pathway in P. aeruginosa but adopts a similar A-T-R domain structure as PvfC.…”