Iron-sulfur (Fe-S) clusters are ubiquitous cofactors in biological systems. Given their central role in bacterial metabolism and pathogenesis, the biogenesis of Fe-S clusters is tightly controlled. We reveal a feedback regulatory mechanism involving the sulfide producing SufS/SufU complex within the sulfur utilization (SUF) system of Mycobacterium tuberculosis , the bacterium that causes tuberculosis. In this mechanism, [2Fe-2S] clusters compete with zinc ions for binding to the sulfide transfer protein SufU. Cluster binding induces SufU tetramerization, which prevents its interaction with the cysteine desulfurase SufS, thereby inhibiting SufS activation and limiting sulfide supply for Fe-S cluster biogenesis. These findings uncover an unrecognized regulatory mechanism in M. tuberculosis , ensuring strict control of Fe-S cluster production.

The ester bond crosslink discovered within bacterial adhesin proteins offers a captivating insight into the convergent evolution of enzyme-like machinery. Crystal structures reveal a putative catalytic triad comprising an acid-base-nucleophile combination and an oxyanion-like site that suggests a serine protease-like mechanism drives the crosslinking process. We now provide confirmation of the mechanism, revealing functional catalytic dyads or triads, and the recapitulation of protease machinery from a Pseudomonas bacterium and a human cytomegalovirus related only by convergent evolution. Molecular dynamics simulations suggest how a conservative threonine-to-serine mutation of the nucleophile induces hydrolysis and eliminates the ester bond crosslink. Collectively, our structural, functional, and computational efforts detail the molecular intricacies of intramolecular ester bond formation and underscore the convergent evolutionary adaptations of bacteria in exploiting enzyme-like machinery to protect essential adhesin proteins from the mechanical, biological, and chemical hostilities of their replicative niche.

We report structures of the Mycobacterium tuberculosis isoprenyl diphosphate synthase Rv2173 in three forms: apo and two substrate-bound forms [isoprenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP)]. The protein possesses a canonical all-α-helical trans-isoprenyl diphosphate synthase fold that is dimeric in each form. There are some differences between the structures: the IPP-bound form shows IPP bound in the DMAPP/allylic substrate-binding site with three divalent metal ions bound around the IPP and the complete C-terminus closing around the active site, while the apo and DMAPP-bound forms are more open, with some of the C-terminal region disordered, supporting suggestions that the C-terminus is important in substrate entry/product exit. In the DMAPP form DMAPP occupies the expected allylic substrate site, but only two metal ions are associated with the binding, with the DMAPP diphosphates adopting a slightly different binding pose compared with IPP in the same site, and the third metal-binding site is unoccupied. In no case is the IPP binding site occupied by IPP. There has been some uncertainty regarding product length for Rv2173, with variable lengths being reported. In the structures reported here, the `capping' residue at the bottom of the binding cavity is tryptophan and comparison with other IPP synthases suggests that the structure of Rv2173 is most consistent with a C10-C15 final product size.

F420-dependent glucose-6-phosphate dehydrogenase (FGD) catalyzes the conversion of glucose-6-phosphate (G6P) to 6-phosphogluconolactone, using cofactor F420 as the hydride transfer acceptor. Our previous pH dependence studies suggested that E109 serves as an active site acid, donating a proton to the N-1 position of F420, while leaving the role of H40 unanswered, which was previously suggested to serve as the active site base. This work utilizes thermodynamic and kinetic studies to elucidate additional mechanistic details concerning the roles of H40 and E13. The E13 residue had not previously been considered as a key player during catalysis. Therefore, the H40A, H40Q, E13A, and E13Q FGD variants were generated and fully characterized to determine their roles in catalysis. Here, we conducted temperature-dependent pH profiles and inactivation experiments using diethylpyrocarbonate (DEPC) to determine the role of H40 during catalysis. The temperature-dependent experiments suggest that an acidic histidine can donate a proton to E13. The inactivation experiments revealed monophasic kinetics, suggesting that the one active site H40 is covalently modified by DEPC. Therefore, the active site base is a deprotonated H40 that abstracts a proton from G6P, and then a hydride is transferred to the C-5 position of cofactor F420. These data suggest that E13 and H40 act as a catalytic dyad. Global analysis of the pre-steady-state experiments revealed the accumulation of an intermediate, the spectrum of which resembles an enzyme-product complex. The global analysis also reveals fast chemistry and slow product release with cofactor association being rate-limiting in catalysis.

Poly-γ-glutamate tails are a distinctive feature of archaeal, bacterial, and eukaryotic cofactors, including the folates and F 420 . Despite decades of research, key mechanistic questions remain as to how enzymes successively add glutamates to poly-γ-glutamate chains while maintaining cofactor specificity. Here, we show how poly-γ-glutamylation of folate and F 420 by folylpolyglutamate synthases and γ-glutamyl ligases, non-homologous enzymes, occurs via processive addition of L -glutamate onto growing γ-glutamyl chain termini. We further reveal structural snapshots of the archaeal γ-glutamyl ligase (CofE) in action, crucially including a bulged-chain product that shows how the cofactor is retained while successive glutamates are added to the chain terminus. This bulging substrate model of processive poly-γ-glutamylation by terminal extension is arguably ubiquitous in such biopolymerisation reactions, including addition to folates, and demonstrates convergent evolution in diverse species from archaea to humans.

Researchers have long sought to `see' proteins and other macromolecules in motion, to better understand their functions. Technological developments, notably advances in serial crystallography, are now making these dreams a reality, heralding a new era of kinetic crystallography.

F420-dependent glucose-6-phosphate dehydrogenase (FGD) is an enzyme found in Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis disease. FGD catalyzes the conversion of glucose-6-phosphate (G6P) to 6-phosphogluconolactone, utilizing an F420 cofactor as a hydride acceptor. This reaction is important in the production of the reduced F420 cofactor and nucleic acid biosynthesis within Mtb, as well as the development of pharmaceuticals to treat multi-drug resistant and extreme drug resistant forms of tuberculosis. Our goal is to understand the reaction mechanism of FGD. An initial proposed mechanism suggests that H40 acted as an active site base, while E109 functioned as the active site acid. Our previous studies confirmed that while E109 does act as the active site acid, H40 does not act as the active site base. To further our investigation, we have generated a series of FGD variants, which include E13A, E13Q, E109A, H40A, H40Q, H260A, H260N to further study H40, while determining the functionality of E13 and H260. We then obtained the binding affinities, steady-state, pre-steady-state kinetic parameters, and pH-rate profiles for wtFGD and the above mentioned FGD variants. The dissociation constant values showed that these amino acids aided in F420 binding but not G6P binding. The steady-state experiments revealed that the residues were important in catalysis due to decreased catalytic activity. The pKa of ionizable groups identified from pH-rate profiles were also investigated in wtFGD as a function of temperature and linearized to the van't Hoff equation to determine standard enthalpy and entropy of active site residue ionization. The results are discussed here. This work was supported by NIH Grant 1R15GM113223-01A1 (to KJW) and the Health Research Council of New Zealand Grant HRC 12/1111 (to E.N.B.).

Cell-surface proteins known as adhesins enable bacteria to colonize particular environments, and in Gram-positive bacteria often contain autocatalytically formed covalent intramolecular cross-links. While investigating the prevalence of such cross-links, a remarkable example was discovered in Mobiluncus mulieris, a pathogen associated with bacterial vaginosis. This organism encodes a putative adhesin of 7651 residues. Crystallography and mass spectrometry of two selected domains, and AlphaFold structure prediction of the remainder of the protein, were used to show that this adhesin belongs to the family of thioester, isopeptide and ester-bond-containing proteins (TIE proteins). It has an N-terminal domain homologous to thioester adhesion domains, followed by 51 immunoglobulin (Ig)-like domains containing ester- or isopeptide-bond cross-links. The energetic cost to the M. mulieris bacterium in retaining such a large adhesin as a single gene or protein construct suggests a critical role in pathogenicity and/or persistence.

This editorial acknowledges the transformative impact of new machine-learning methods, such as the use of AlphaFold, but also makes the case for the continuing need for experimental structural biology.

Group A Streptococcus (GAS) is a globally important pathogen causing a broad range of human diseases. GAS pili are elongated proteins with a backbone comprised repeating T-antigen subunits, which extend from the cell surface and have important roles in adhesion and establishing infection. No GAS vaccines are currently available, but T-antigen-based candidates are in pre-clinical development. This study investigated antibody-T-antigen interactions to gain molecular insight into functional antibody responses to GAS pili. Large, chimeric mouse/human Fab-phage libraries generated from mice vaccinated with the complete T18.1 pilus were screened against recombinant T18.1, a representative two-domain T-antigen. Of the two Fab identified for further characterization, one (designated E3) was cross-reactive and also recognized T3.2 and T13, while the other (H3) was type-specific reacting with only T18.1/T18.2 within a T-antigen panel representative of the major GAS T-types. The epitopes for the two Fab, determined by x-ray crystallography and peptide tiling, overlapped and mapped to the N-terminal region of the T18.1 N-domain. This region is predicted to be buried in the polymerized pilus by the C-domain of the next T-antigen subunit. However, flow cytometry and opsonophagocytic assays showed that these epitopes were accessible in the polymerized pilus at 37°C, though not at lower temperature. This suggests that there is motion within the pilus at physiological temperature, with structural analysis of a covalently linked T18.1 dimer indicating "knee-joint" like bending occurs between T-antigen subunits to expose this immunodominant region. This temperature dependent, mechanistic flexing provides new insight into how antibodies interact with T-antigens during infection.

A recently-validated and underexplored drug target in Mycobacterium tuberculosis is PptT, an essential phosphopantetheinyl transferase (PPTase) that plays a critical role in activating enzymes for both primary and secondary metabolism. PptT possesses a deep binding pocket that does not readily accept labelled coenzyme A analogues that have previously been used to screen for PPTase inhibitors. Here we report on the development of a high throughput, colourimetric screen that monitors the PptT-mediated activation of the non-ribosomal peptide synthetase BpsA to a blue pigment (indigoidine) synthesising form in vitro. This screen uses unadulterated coenzyme A, avoiding analogues that may interfere with inhibitor binding, and requires only a single-endpoint measurement. We benchmark the screen using the well-characterised Library of Pharmaceutically Active Compounds (LOPAC(1280)) collection and show that it is both sensitive and able to distinguish weak from strong inhibitors. We further show that the BpsA assay can be applied to quantify the level of inhibition and generate consistent EC50 data. We anticipate these tools will facilitate both the screening of established chemical collections to identify new anti-mycobacterial drug leads and to guide the exploration of structure-activity landscapes to improve existing PPTase inhibitors.

F420-dependent Glucose-6-phosphate Dehydrogenase (FGD) is found within Mycobacterium tuberculosis, the causative agent of tuberculosis disease (TB). FGD catalyzes the conversion of glucose-6-phosphate (G6P) to 6-phosphogluconolactone using the oxidized F420 cofactor, which becomes reduced during catalysis (Figure 1). Previous crystallographic studies on wild-type FGD suggested that conserved residues, H40 and Glu 109 acted as the active site base and active site acid, respectively. However, our previous pH profile experiments suggested that while Glu 109 does act as the acid, His 40 does not act as the active site base. Our present work focuses on determining which active site residue could act as the active site base. For this reason, we have created FGD variants of Glu13,, His 260 and H40. We have conducted kinetic isotope effect experiments as well as pH dependence studies in order to elucidate their roles during catalysis. Our results will be discussed here.