SummaryTonB couples the cytoplasmic membrane protonmotive force (pmf) to active transport across the outer membrane, potentially through a series of conformational changes. Previous studies of a TonB transmembrane domain mutant (TonB-⌬V17) and its phenotypical suppressor (ExbB-A39E) suggested that TonB is conformationally sensitive. Here, two new mutations of the conserved TonB transmembrane domain SHLS motif were isolated, TonB-S16L and -H20Y, as were two new suppressors, ExbB-V35E and -V36D. Each suppressor ExbB restored at least partial function to the TonB mutants, although TonB-⌬V17, for which both the conserved motif and the register of the predicted transmembrane domain ␣-helix are affected, was the most refractory. As demonstrated previously, TonB can undergo at least one conformational change, provided both ExbB and a functional TonB transmembrane domain are present. Here, we show that this conformational change reflects the ability of TonB to respond to the cytoplasmic membrane proton gradient, and occurs in proportion to the level of TonB activity attained by mutant-suppressor pairs. The phenotype of TonB-⌬V17 was more complex than the -S16L and -H20Y mutations, in that, beyond the inability to be energized efficiently, it was also conditionally unstable. This second defect was evident only after suppression by the ExbB mutants, which allow transmembrane domain mutants to be energized, and presented as the rapid turnover of TonB-⌬V17. Importantly, this degradation was dependent upon the presence of a TonB-dependent ligand, suggesting that TonB conformation also changes following the energy transduction event. Together, these observations support a dynamic model of energy transduction in which TonB cycles through a set of conformations that differ in potential energy, with a transition to a higher energy state driven by pmf and a transition to a lower energy state accompanying release of stored potential energy to an outer membrane receptor.

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Natural products biosynthesized wholly or in part by nonribosomal peptide synthetases (NRPSs) are some of the most important drugs currently used clinically for the treatment of a variety of diseases. Since the initial research into NRPSs in the early 1960s, we have gained considerable insights into the mechanism by which these enzymes assemble these natural products. This review will present a brief history of how the basic mechanistic steps of NRPSs were initially deciphered and how this information has led us to understand how nature modified these systems to generate the enormous structural diversity seen in nonribosomal peptides. This review will also briefly discuss how drug development and discovery are being influenced by what we have learned from nature about nonribosomal peptide biosynthesis.

The biosynthesis of many natural products of clinical interest involves large, multi-domain enzymes called nonribosomal peptide synthetases (NRPSs). In bacteria, many of the gene clusters coding for NRPSs also code for a member of the MbtH-like protein superfamily, which are small proteins of unknown function. Using MbtH-like proteins from three separate NRPS systems, we show that these proteins co-purify together with the NRPSs and influence amino acid activation. As a consequence, MbtH-like proteins are integral components of NRPSs.Nonribosomal peptide synthetases (NRPSs) are involved in the assembly of natural products of clinical interest such as the antibacterial drugs vancomycin, daptomycin, and capreomycin. A basic understanding how NRPSs catalyze the assembly of such molecules from simple precursors has been established (1). During assembly, each precursor is activated, covalently tethered to the NRPS, and then directionally condensed into the growing molecule by a set of catalytic domains grouped together as modules. Each module is typically composed of an adenylation (A) domain that recognizes and activates each precursor and tethers them to a peptidyl carrier protein (PCP) domain as a thioester. Condensation (C) domains subsequently catalyze directional bond formation between two PCP-linked precursors. Additional domains can add functionality to the precursors or govern its release from the NRPS. The repeating domain/modular structure of NRPSs provides an assembly line-like logic to the biosynthesis of the associated natural products.In bacteria, many gene clusters coding for the NRPS involved in the production of natural products also code for a small (∼70 amino acid) protein containing three conserved tryptophan residues. These proteins have been named the MbtH-like protein superfamily based on their similarity to MbtH from the mycobactin biosynthesis gene cluster (2). The production of some NRPS-dependent natural products requires an MbtH-like protein (3,4), but how these proteins influence production is unknown. Co-production of an MbtH-like protein with an NRPS component enhances protein production levels (5). A direct role in catalysis has been questioned by a report that the enterobactin (ENT) NRPS is functional in vitro in the absence of the associated MbtH-like protein (6). Structural work on the MbtHlike protein from the pyroverdine system (3) and MbtH itself (7) did not reveal any motifs suggestive of a catalytic site, instead, a role in protein-protein interactions.We are investigating the biosynthesis of the antituberculosis drugs capreomycin (CMN) and viomycin (VIO) to better understand NRPS enzymology and develop new derivatives of these drugs using combinatorial biosynthesis. These structurally related non-ribosomal * To whom correspondence should be addressed. Phone: (608) Figure S1, Supporting Information) (8, 9). In addition to the NRPS components, the associated gene clusters code for MbtH-like proteins. This provided us with two related NRPS systems to address questions co...

Visceral leishmaniasis (VL), caused by the protozoan parasites Leishmania donovani and Leishmania infantum, is one of the major parasitic diseases worldwide. There is an urgent need for new drugs to treat VL, because current therapies are unfit for purpose in a resource-poor setting. Here, we describe the development of a preclinical drug candidate, GSK3494245/DDD01305143/compound 8, with potential to treat this neglected tropical disease. The compound series was discovered by repurposing hits from a screen against the related parasite Trypanosoma cruzi. Subsequent optimization of the chemical series resulted in the development of a potent cidal compound with activity against a range of clinically relevant L. donovani and L. infantum isolates. Compound 8 demonstrates promising pharmacokinetic properties and impressive in vivo efficacy in our mouse model of infection comparable with those of the current oral antileishmanial miltefosine. Detailed mode of action studies confirm that this compound acts principally by inhibition of the chymotrypsin-like activity catalyzed by the β5 subunit of the L. donovani proteasome. High-resolution cryo-EM structures of apo and compound 8-bound Leishmania tarentolae 20S proteasome reveal a previously undiscovered inhibitor site that lies between the β4 and β5 proteasome subunits. This induced pocket exploits β4 residues that are divergent between humans and kinetoplastid parasites and is consistent with all of our experimental and mutagenesis data. As a result of these comprehensive studies and due to a favorable developability and safety profile, compound 8 is being advanced toward human clinical trials.