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BIOSYNTHESIS OF RIBOSOMALLY SYNTHESIZED ANTIBIOTICS

A large group of bacterial natural products are produced by enzymatic transformations of ribosomally-synthesized precursors. Such compounds are termed "RiPPs" for ribosomally-synthesized post-translationally modified peptides. Some RiPPs are antibiotics, others could be virulence factors ore even provide chemical defense against phages.

RiPP biosynthetic enzymes assemble into large complexes that could introduce peptide backbone modifications in RiPPs: conversion of amino acids into heterocycles, thioamidation (replacement of peptide bond oxygen by sulfur), or even folding peptides in 3D threaded lasso structures. In principle, RiPP proteins are capable of modifying virtually any substrate peptide bearing a correct recognition sequence, making them highly valuable for industry. However, we lack a good understanding of how these systems work and how they could be engineered.

We work with microcin B17, klebsazolicin, streptolysin S and lasso peptides RiPP systems to unravel the mechanistic details of how the biosynthetic systems assembly and function.

DNA TOPOISOMERASES & NEW ANTIBIOTICS
Bacterial type II topoisomerases gyrase and topoisomerase IV are essential bacterial enzymes, and important antibiotic targets. They work in close connection with the replisome and SMC proteins, directly and indirectly affecting all genomic transactions in the cell. The research in the lab aims to address a fundamental question of how topoisomerases use energy of ATP to introduce defined topology in DNA. We have developed cryoEM approaches to study gyrase and topoisomerase IV, and recently determined a first structure of gyrase with a chirally wrapped DNA loop. We are working on developing various approaches of imaging gyrase and topoisomerase IV with topologically closed substrates such as DNA rings of various size, and on time-resolved cryoEM to dissect the details of the catalytic mechanism.

DNA GYRASE & FLUOROQUINOLONE RESISTANCE

DNA gyrase inhibitors fluoroquinolones are one of the most prescribed antibiotics. Bacterial genomes encode special proteins to endow gyrase with a degree of resistance to these drugs. We are using cryo-EM to study molecular mechanisms of this process, which involves binding of proteins such as QnrB1 or MfpA to DNA gyrase, resulting in the release of the drug. In addition, we are working on understanding DNA repair mechanisms responsible for the eliminating topoisomerase-DNA crosslinks caused by fluoroquinolones on molecular and cellular level, and ultimately for the differences in antibiotics lethality.

BACTERIAL DEFENSE SYSTEMS

An enormous diversity of bacterial anti-phage defense systems, and a parallel diversity of phage-encoded small protein inhibitors of these systems is one of the most consequential findings of the recent years. Phage defense systems provide many fascinating examples of molecular machines, and frequently utilise the same domains as topoisomerases for example TOPRIM or GHKL domains. Together with our collaborators, we are investigating molecular mechanism of on the most widespread but mysterious multi-component system BREX (bacteriophage exclusion system) and have recently determined structures of BREX methyltransferase BrxX in complex with substrate DNA and with a phage-encoded DNA mimic inhibitor Ocr.

ANTIBIOTIC TRANSPORT

To make an effective drug, we need to know how to deliver it to the target cell.
Bacterial outer and inner membrane transporters are key to the sensitivity to antibacterial peptides. In collaboration with the groups of Konstantinos Beis, Christos Pliotas and Jonathan Heddle we study the molecular mechanisms of antibiotic transport and determinants for the compound selectivity. We use lipid membrane mimetics (nanodiscs) and cryo-electron microscopy to look at the transporters in the conditions most close to native.