Mechanistic analysis of nonribosomal peptide synthetases

Considering the ongoing rise of the multidrug-resistant bacterial infections, it is essential to expand the available repertoire of therapeutic agents. Microbial natural products are an indispensable source of novel activities and continue to serve as our main provider of antibiotics and chemotherapeutics. Nonribosomal peptides are among the most widespread natural products in bacteria and fungi. Their importance is best illustrated by their complexity and the amounts of resources dedicated to building the underlying biosynthetic machineries nonribosomal peptide synthetases (NRPS). These gigantic, multidomain enzymes synthesize peptides by linking individual amino acid units in an assembly line fashion. Six decades of NRPS research have resulted in several remarkable tailoring successes. However, the lack of mechanistic understanding of the inner workings of NRPSs has prevented the development of a general workflow which would reliably generate functional enzymes and new drugs. Aspiring to alleviate these obstacles, this thesis offers critical insights into adenylation and the interplay with condensation, two fundamental NRPS reactions

Sulfonium Acids Loaded onto an Unusual Thiotemplate Assembly Line Construct the Cyclopropanol Warhead of a Burkholderia Virulence Factor

Pathogenic bacteria of the Burkholderia pseudomallei group cause severe infectious diseases such as glanders and melioidosis. Malleicyprols were identified as important bacterial virulence factors, yet the biosynthetic origin of their cyclopropanol warhead has remained enigmatic. By a combination of mutational analysis and metabolomics we found that sulfonium acids, dimethylsulfoniumpropionate (DMSP) and gonyol, known as osmolytes and as crucial components in the global organosulfur cycle, are key intermediates en route to the cyclopropanol unit. Functional genetics and in vitro analyses uncover a specialized pathway to DMSP involving a rare prokaryotic SET-domain methyltransferase for a cryptic methylation, and show that DMSP is loaded onto the NRPS-PKS hybrid assembly line by an adenylation domain dedicated to zwitterionic starter units. Then, the megasynthase transforms DMSP into gonyol, as demonstrated by heterologous pathway reconstitution in E. coli. © 2020 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA

Total Synthesis and Functional Evaluation of IORs, Sulfonolipid‐based Inhibitors of Cell Differentiation in Salpingoeca rosetta

The choanoflagellate Salpingoeca rosetta is an important model system to study the evolution of multicellularity. In this study we developed a new, modular, and scalable synthesis of sulfonolipid IOR‐1A (six steps, 27 % overall yield), which acts as bacterial inhibitor of rosette formation in S. rosetta . The synthesis features a decarboxylative cross‐coupling reaction of a sulfonic acid‐containing tartaric acid derivative with alkyl zinc reagents. Synthesis of 15 modified IOR‐1A derivatives, including fluorescent and photoaffinity‐based probes, allowed quantification of IOR‐1A, localization studies within S. rosetta cells, and evaluation of structure‐activity relations. In a proof of concept study, an inhibitory bifunctional probe was employed in proteomic profiling studies, which allowed to deduce binding partners in bacteria and S. rosetta . These results showcase the power of synthetic chemistry to decipher the biochemical basis of cell differentiation processes within S. rosetta

An Engineered Nonribosomal Peptide Synthetase Shows Opposite Amino Acid Loading and Condensation Specificity

Engineering of nonribosomal peptide synthetases (NRPS) has faced numerous obstacles despite being an attractive path towards novel bioactive molecules. Specificity filters in the nonribosomal peptide assembly line determine engineering success, but the relative contribution of adenylation (A-) and condensation (C-)domains is under debate. In the engineered, bimodular NRPS sdV-GrsA/GrsB1, the first module is a subdomain-swapped chimera showing substrate promiscuity. On sdV-GrsA and evolved mutants, we have employed kinetic modelling to investigate product specificity under substrate competition. Our model contains one step, in which the A-domain acylates the thiolation (T-)domain, and one condensation step deacylating the T-domain. The simplified model agrees well with experimentally determined acylation preferences and shows that the condensation specificity is mismatched with the engineered acylation specificity. Our model predicts changing product specificity in the course of the reaction due to dynamic T-domain loading, and that A-domain overrules C-domain specificity when T-domain loading reaches a steady-state. Thus, we have established a tool for investigating poorly accessible C-domain specificity through nonlinear kinetic modeling and gained critical insights how the interplay of A- and C-domains determines the product specificity of NRPSs. </div