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...

Active transport of vitamin B12 and Fe(III)-siderophore complexes across the outer membrane of Escherichia coli appears to be dependent upon the ability of the TonB protein to couple cytoplasmic membrane-generated protonmotive force to outer membrane receptors. TonB is supported in this role by an auxiliary protein, ExbB, which, in addition to stabilizing TonB against the activities of endogenous envelope proteases, directly contributes to the energy transduction process. The topological partitioning of TonB and ExbB to either side of the cytoplasmic membrane restricts the sites of interaction between these proteins primarily to their transmembrane domains. In this study, deletion of valine 17 within the aminoterminal transmembrane anchor of TonB resulted in complete loss of TonB activity, as well as loss of detectable in vivo crosslinking into a 59 kDa complex believed to contain ExbB. The delta V17 mutation had no effect on TonB export. The loss of crosslinking appeared to reflect conformational changes in the TonB/ExbB pair rather than loss of interaction since ExbB was still required for some stabilization of TonB delta V17. Molecular modeling suggested that the delta V17 mutation caused a significant change in the predicted conserved face of the TonB amino-terminal membrane anchor. TonB delta V17 was unable to achieve the 23 kDa proteinase K-resistant form in lysed sphaeroplasts that is characteristic of active TonB. Wild-type TonB also failed to achieve the proteinase K-resistant configuration when ExbB was absent. Taken together these results suggested that the delta V17 mutation interrupted productive TonB-ExbB interactions. The apparent ability to crosslink to ExbB as well as a limited ability to transduce energy were restored by a second mutation (A39E) in or near the first predicted transmembrane domain of the ExbB protein. Consistent with the weak suppression, a 23 kDa proteinase K-resistant form of TonB delta V17 was not observed in the presence of ExbBA39E. Neither the ExbBA39E allele nor the absence of ExbB affected TonB or TonB delta V17 export. Unlike the tonB delta V17 mutation, the exbBA39E mutation did not greatly alter a modelled ExbB transmembrane domain structure. Furthermore, the suppressor ExbBA39E functioned normally with wild-type TonB, suggesting that the suppressor was not allele specific. Contrary to expectations, the TonB delta V17, ExbBA39E pair resulted in a TonB with a greatly reduced half-life (approximately 10 min). These results together with protease susceptibility studies suggest that ExbB functions by modulating the conformation of TonB.

The tuberactinomycin antibiotics are essential components in the drug arsenal against Mycobacterium tuberculosis infections and are specifically used for the treatment of multidrug-resistant tuberculosis. These antibiotics are also being investigated for their targeting of the catalytic RNAs involved in viral replication and for the treatment of bacterial infections caused by methicillin-resistant Staphylococcus aureus strains and vancomycin-resistant enterococci. We report on the isolation, sequencing, and annotation of the biosynthetic gene cluster for one member of this antibiotic family, viomycin, from Streptomyces sp. strain ATCC 11861. This is the first gene cluster for a member of the tuberactinomycin family of antibiotics sequenced, and the information gained can be extrapolated to all members of this family. The gene cluster covers 36.3 kb of DNA and encodes 20 open reading frames that we propose are involved in the biosynthesis, regulation, export, and activation of viomycin, in addition to self-resistance to the antibiotic. These results enable us to predict the metabolic logic of tuberactinomycin production and begin steps toward the combinatorial biosynthesis of these antibiotics to complement existing chemical modification techniques to produce novel tuberactinomycin derivatives.It was recently estimated that between the years 1998 and 2030 there will be 225 million new cases of tuberculosis (TB) and 79 million TB-related deaths (40). These numbers are astonishing when one considers that treatments for this disease, in the forms of vaccines or chemotherapy, have been available for more than 50 years (29). Mycobacterium tuberculosis, the causative agent of TB, is notoriously slowly growing and during infection can persist in a latent form in many individuals. These attributes contribute to the reasons why typical chemotherapy regimens for TB last 6 to 9 months (6) and why TB is so persistent. This prolonged treatment presents significant hurdles in the development of new antibiotics and in retaining the efficacies of the antibiotics used at present. Side effects and toxicity from a particular compound can be magnified when a patient takes a drug for this length of time, and there are increased incidences of poor adherence to the chemotherapy regimen by unmonitored patients, resulting in the development of multidrug-resistant (MDR) TB infections. These facts, together with alarming interactions between human immunodeficiency virus and TB infections that can result in increased numbers of infected individuals and MDR TB (30), make it of paramount importance to develop new chemotherapy agents or introduce modifications to the agents available at present to reduce their toxicities and increase their activities against MDR TB.The tuberactinomycins (TUBs; this abbreviation refers to the antibiotic family as a whole) (Fig. 1) are used specifically for the treatment of MDR TB (14). The importance of TUBs is reflected by some members being included on the World Health Organization's Model List of Essential M...

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.

The glycopeptide antibiotics vancomycin and teicoplanin are vital components of modern anti-infective chemotherapy exhibiting outstanding activity against Gram-positive pathogens including members of the genera Streptococcus, Staphylococcus, and Enterococcus. These antibiotics also provide fascinating examples of the chemical and associated biosynthetic complexity exploitable in the synthesis of natural products by actinomycetes group of bacteria. We report the sequencing and annotation of the biosynthetic gene cluster for the glycopeptide antibiotic A47934 from Streptomyces toyocaensis NRRL15009, the first complete sequence for a teicoplanin class glycopeptide. The cluster includes 34 ORFs encompassing 68 kb and includes all of the genes predicted to be required to synthesize A47934 and regulate its biosynthesis. The gene cluster also contains ORFs encoding enzymes responsible for glycopeptide resistance. This role was confirmed by insertional inactivation of the D-Ala-D-lactate ligase, vanAst, which resulted in the predicted A47934-sensitive phenotype and impaired antibiotic biosynthesis. These results provide increased understanding of the biosynthesis of these complex natural products.G lycopeptide antibiotics (GPAs) have been mainstays of antimicrobial chemotherapy since their discovery in the mid 1950s. These antibiotics act exclusively on Gram-positive bacteria by forming a tight and specific noncovalent complex with the D-Ala-D-Ala terminus of the peptidoglycan, inhibiting cell wall growth and crosslinking (1, 2). Clinical resistance to GPAs was first described in the enterococci in 1988 (3), and resistance has now manifested itself in the more virulent streptococci and staphylococci (reviewed in ref. 4). GPA resistance is now a significant worldwide phenomenon that has severely impacted the health care sector both in increased mortality and morbidity, and economically (5). The predominant mechanism of resistance is the synthesis of cell wall peptidoglycan terminating in D-Ala-D-lactate, which dramatically decreases the affinity of these antibiotics for their target (6).GPAs are exclusively obtained through fermentation, yet despite their importance, their biosynthesis is not well understood. They are comprised of a heptapeptide core consisting of both common and unusual amino acids (4). Crosslinking of the amino acids through aryl ether and carbon-carbon bonds provides rigidity to the peptide. There are two major structural classes of GPAs based on the identity of the core peptide, and the two clinically used GPAs, vancomycin and teicoplanin, exemplify both classes (Fig. 1). An additional class of structurally homologous secondary metabolites with anticomplement activity is exemplified by complestatin (Fig. 1). Structural diversity in these natural products is achieved through changes in the peptide backbone, and selective amino acid halogenation, glycosylation, lipidation, methylation, and sulfonylation. In principle, understanding of the genetic and mechanistic basis of these modifications could lead ...