Teixobactin and its analogues: a new hope in antibiotic discovery

Increasing bacterial resistance against current antibiotics and lack of new molecules to combat bacterial resistance are key challenges to global health. There is, therefore, a continuing need to develop new antibiotics. Teixobactin, a cyclic undecapeptide, displays excellent antibacterial activities against a range of pathogenic bacteria, such as methicillin-resistant Staphylococcus aureus (MRSA) and Mycobacterium tuberculosis. Interestingly, it operates by multiple modes of actions and is bactericidal toward S. aureus without detectable resistance. This unique combination of wide Gram-positive activity coupled with its inability to elicit resistance make teixobactin a very attractive molecule for antimicrobial therapeutic development. This Viewpoint discusses teixobactin, its analogues, and the challenges and opportunities associated with their future development

Vinylogous Dehydration by a Polyketide Dehydratase Domain in Curacin Biosynthesis

Polyketide synthase (PKS) enzymes continue to hold great promise as synthetic biology platforms for the production of novel therapeutic agents, biofuels, and commodity chemicals. Dehydratase (DH) catalytic domains play an important role during poly­ketide biosynthesis through the dehydration of the nascent poly­ketide intermediate to provide olefins. Our understanding of the detailed mechanistic and structural underpinning of DH domains that control substrate specificity and selectivity remains limited, thus hindering our efforts to rationally re-engineer PKSs. The curacin pathway houses a rare plurality of possible double bond permutations containing conjugated olefins as well as both <i>cis</i>- and <i>trans</i>-olefins, providing an unrivaled model system for poly­ketide dehydration. All four DH domains implicated in curacin biosynthesis were characterized <i>in vitro</i> using synthetic substrates, and activity was measured by LC-MS/MS analysis. These studies resulted in complete kinetic characterization of the <i>all-trans</i>-trienoate-forming CurK-DH, whose <i>k</i>_cat of 72 s^–1 is more than 3 orders of magnitude greater than that of any previously reported PKS DH domain. A novel stereospecific mechanism for diene formation involving a vinylogous enolate intermediate is proposed for the CurJ and CurH DHs on the basis of incubation studies with truncated substrates. A synthetic substrate was co-crystallized with a catalytically inactive Phe substitution in the His-Asp catalytic dyad of CurJ-DH to elucidate substrate–enzyme interactions. The resulting complex suggested the structural basis for dienoate formation and provided the first glimpse into the enzyme–substrate interactions essential for the formation of olefins in poly­ketide natural products. This examination of both canonical and non-canonical dehydration mechanisms reveals hidden catalytic activity inherent in some DH domains that may be leveraged for future applications in synthetic biology

Domain Organization and Active Site Architecture of a Polyketide Synthase <i>C</i>‑methyltransferase

Polyketide metabolites produced by modular type I polyketide synthases (PKS) acquire their chemical diversity through the variety of catalytic domains within modules of the pathway. Methyltransferases are among the least characterized of the catalytic domains common to PKS systems. We determined the domain boundaries and characterized the activity of a PKS <i>C</i>-methyltransferase (<i>C</i>-MT) from the curacin A biosynthetic pathway. The <i>C</i>-MT catalyzes <i>S</i>-adenosylmethionine-dependent methyl transfer to the α-position of β-ketoacyl substrates linked to acyl carrier protein (ACP) or a small-molecule analog but does not act on β-hydroxyacyl substrates or malonyl-ACP. Key catalytic residues conserved in both bacterial and fungal PKS <i>C</i>-MTs were identified in a 2 Å crystal structure and validated biochemically. Analysis of the structure and the sequences bordering the <i>C</i>-MT provides insight into the positioning of this domain within complete PKS modules

Structural Basis of Polyketide Synthase O‑Methylation

Modular type I polyketide synthases (PKSs) produce some of the most chemically complex metabolites in nature through a series of multienzyme modules. Each module contains a variety of catalytic domains to selectively tailor the growing molecule. PKS O-methyltransferases ( O-MTs) are predicted to methylate β-hydroxyl or β-keto groups, but their activity and structure have not been reported. We determined the domain boundaries and characterized the catalytic activity and structure of the StiD and StiE O-MTs, which methylate opposite β-hydroxyl stereocenters in the myxobacterial stigmatellin biosynthetic pathway. Substrate stereospecificity was demonstrated for the StiD O-MT. Key catalytic residues were identified in the crystal structures and investigated in StiE O-MT via site-directed mutagenesis and further validated with the cyanobacterial CurL O-MT from the curacin biosynthetic pathway. Initial structural and biochemical analysis of PKS O-MTs supplies a new chemoenzymatic tool, with the unique ability to selectively modify hydroxyl groups during polyketide biosynthesis

Polyketide Intermediate Mimics as Probes for Revealing Cryptic Stereochemistry of Ketoreductase Domains

Among natural product families, polyketides have shown the most promise for combinatorial biosynthesis of natural product-like libraries. Though recent research in the area has provided many mechanistic revelations, a basic-level understanding of kinetic and substrate tolerability is still needed before the full potential of combinatorial biosynthesis can be realized. We have developed a novel set of chemical probes for the study of ketoreductase domains of polyketide synthases. This chemical tool-based approach was validated using the ketoreductase of pikromycin module 2 (PikKR2) as a model system. Triketide substrate mimics <b>12</b> and <b>13</b> were designed to increase stability (incorporating a nonhydrolyzable thioether linkage) and minimize nonessential functionality (truncating the phosphopantetheinyl arm). PikKR2 reduction product identities as well as steady-state kinetic parameters were determined by a combination of LC-MS/MS analysis of synthetic standards and a NADPH consumption assay. The d-hydroxyl product is consistent with bioinformatic analysis and results from a complementary biochemical and molecular biological approach. When compared to widely employed substrates in previous studies, diketide <b>63</b> and <i>trans</i>-decalone <b>64</b>, substrates <b>12</b> and <b>13</b> showed 2–10 fold lower <i>K</i>_M values (2.4 ± 0.8 and 7.8 ± 2.7 mM, respectively), indicating molecular recognition of intermediate-like substrates. Due to an abundance of the nonreducable enol-tautomer, the <i>k</i>_cat values were attenuated by as much as 15–336 fold relative to known substrates. This study reveals the high stereoselectivity of PikKR2 in the face of gross substrate permutation, highlighting the utility of a chemical probe-based approach in the study of polyketide ketoreductases

Functional Characterization of a Dehydratase Domain from the Pikromycin Polyketide Synthase

Metabolic engineering of polyketide synthase (PKS) pathways represents a promising approach to natural products discovery. The dehydratase (DH) domains of PKSs, which generate an α,β-unsaturated bond through a dehydration reaction, have been poorly studied compared with other domains, likely because of the simple nature of the chemical reaction they catalyze and the lack of a convenient assay to measure substrate turnover. Herein we report the first steady-state kinetic analysis of a PKS DH domain employing LC–MS/MS analysis for product quantitation. PikDH2 was selected as a model DH domain. Its substrate specificity and mechanism were interrogated with a systematic series of synthetic triketide substrates containing a nonhydrolyzable thioether linkage as well as by site-directed mutagenesis, evaluation of the pH dependence of the catalytic efficiency (<i>V</i>_max/<i>K</i>_M), and kinetic characterization of a mechanism-based inhibitor. These studies revealed that PikDH2 converts d-alcohol substrates to <i>trans</i>-olefin products. The reaction is reversible with equilibrium constants ranging from 1.2 to 2. Moreover, the enzyme activity is robust, and PikDH2 was used on a preparative scale for the chemoenzymatic synthesis of unsaturated triketide products. PikDH2 was shown to possess remarkably strict substrate specificity and is unable to turn over substrates that are epimeric at the β-, γ-, or δ-position. We also demonstrated that PikDH2 has a key ionizable group with a p<i>K</i>_a of 7.0 and can be irreversibly inactivated through covalent modification by a mechanism-based inhibitor, which provides a foundation for future structural studies to elucidate substrate–protein interactions

Immune regulation by fungal strain diversity in inflammatory bowel disease

The fungal microbiota (mycobiota) is an integral part of the complex multi-kingdom microbial community colonizing the mammalian gastrointestinal tract and plays an important role in immune regulation(1–6). Although aberrant mycobiota changes have been linked to several diseases including inflammatory bowel disease (IBD)(3–9), it is currently unknown whether fungal species captured by deep sequencing represent living organisms and whether specific fungi have functional consequences for disease development in affected individuals. Here we developed a translational platform for the functional exploration of the mycobiome at a fungal strain- and patient-specific level. Combining high-resolution mycobiota-sequencing, fungal culturomics and genomics, CRISPR/Cas9-based fungal strain editing system, in vitro functional immunoreactivity assays and in vivo models, this platform allows to explore host-fungal crosstalk within the human gut. We discovered a rich genetic diversity of opportunistic Candida albicans strains that dominated the colonic mucosa of IBD patients. Among these human gut-derived isolates, strains with high immune cell-damaging capacity (HD strains) reflect disease features of individual ulcerative colitis patients and aggravated intestinal inflammation in vivo through IL-1β-dependent mechanisms. Niche-specific inflammatory immunity and Th17 antifungal responses by HD strains in the gut were dependent upon the C. albicans secreted peptide toxin candidalysin during the transition from a benign commensal to a pathobiont state. These findings unveil the strain-specific nature of host-fungal interactions in the human gut and highlight new diagnostic and therapeutic targets for diseases of inflammatory origin