Transition transferases prime bacterial capsule polymerization.

Capsules are long-chain carbohydrate polymers that envelop the surfaces of many bacteria, protecting them from host immune responses. Capsule biosynthesis enzymes are potential drug targets and valuable biotechnological tools for generating vaccine antigens. Despite their importance, it remains unknown how structurally variable capsule polymers of Gram-negative pathogens are linked to the conserved glycolipid anchoring these virulence factors to the bacterial membrane. Using Actinobacillus pleuropneumoniae as an example, we demonstrate that CpsA and CpsC generate a poly(glycerol-3-phosphate) linker to connect the glycolipid with capsules containing poly(galactosylglycerol-phosphate) backbones. We reconstruct the entire capsule biosynthesis pathway in A. pleuropneumoniae serotypes 3 and 7, solve the X-ray crystal structure of the capsule polymerase CpsD, identify its tetratricopeptide repeat domain as essential for elongating poly(glycerol-3-phosphate) and show that CpsA and CpsC stimulate CpsD to produce longer polymers. We identify the CpsA and CpsC product as a wall teichoic acid homolog, demonstrating similarity between the biosynthesis of Gram-positive wall teichoic acid and Gram-negative capsules.

Architecture, Function, Regulation, and Evolution of α-Glucans Metabolic Enzymes in Prokaryotes.

Bacteria have acquired sophisticated mechanisms for assembling and disassembling polysaccharides of different chemistry. α-d-Glucose homopolysaccharides, so-called α-glucans, are the most widespread polymers in nature being key components of microorganisms. Glycogen functions as an intracellular energy storage while some bacteria also produce extracellular assorted α-glucans. The classical bacterial glycogen metabolic pathway comprises the action of ADP-glucose pyrophosphorylase and glycogen synthase, whereas extracellular α-glucans are mostly related to peripheral enzymes dependent on sucrose. An alternative pathway of glycogen biosynthesis, operating via a maltose 1-phosphate polymerizing enzyme, displays an essential wiring with the trehalose metabolism to interconvert disaccharides into polysaccharides. Furthermore, some bacteria show a connection of intracellular glycogen metabolism with the genesis of extracellular capsular α-glucans, revealing a relationship between the storage and structural function of these compounds. Altogether, the current picture shows that bacteria have evolved an intricate α-glucan metabolism that ultimately relies on the evolution of a specific enzymatic machinery. The structural landscape of these enzymes exposes a limited number of core catalytic folds handling many different chemical reactions. In this Review, we present a rationale to explain how the chemical diversity of α-glucans emerged from these systems, highlighting the underlying structural evolution of the enzymes driving α-glucan bacterial metabolism.

A multi-enzyme machine polymerizes the Haemophilus influenzae type b capsule.

Bacterial capsules have critical roles in host-pathogen interactions. They provide a protective envelope against host recognition, leading to immune evasion and bacterial survival. Here we define the capsule biosynthesis pathway of Haemophilus influenzae serotype b (Hib), a Gram-negative bacterium that causes severe infections in infants and children. Reconstitution of this pathway enabled the fermentation-free production of Hib vaccine antigens starting from widely available precursors and detailed characterization of the enzymatic machinery. The X-ray crystal structure of the capsule polymerase Bcs3 reveals a multi-enzyme machine adopting a basket-like shape that creates a protected environment for the synthesis of the complex Hib polymer. This architecture is commonly exploited for surface glycan synthesis by both Gram-negative and Gram-positive pathogens. Supported by biochemical studies and comprehensive 2D nuclear magnetic resonance, our data explain how the ribofuranosyltransferase CriT, the phosphatase CrpP, the ribitol-phosphate transferase CroT and a polymer-binding domain function as a unique multi-enzyme assembly.

Mechanism of high-mannose N-glycan breakdown and metabolism by Bifidobacterium longum.

Bifidobacteria are early colonizers of the human gut and play central roles in human health and metabolism. To thrive in this competitive niche, these bacteria evolved the capacity to use complex carbohydrates, including mammalian N-glycans. Herein, we elucidated pivotal biochemical steps involved in high-mannose N-glycan utilization by Bifidobacterium longum. After N-glycan release by an endo-ß-N-acetylglucosaminidase, the mannosyl arms are trimmed by the cooperative action of three functionally distinct glycoside hydrolase 38 (GH38) α-mannosidases and a specific GH125 α-1,6-mannosidase. High-resolution cryo-electron microscopy structures revealed that bifidobacterial GH38 α-mannosidases form homotetramers, with the N-terminal jelly roll domain contributing to substrate selectivity. Additionally, an α-glucosidase enables the processing of monoglucosylated N-glycans. Notably, the main degradation product, mannose, is isomerized into fructose before phosphorylation, an unconventional metabolic route connecting it to the bifid shunt pathway. These findings shed light on key molecular mechanisms used by bifidobacteria to use high-mannose N-glycans, a perennial carbon and energy source in the intestinal lumen.

SigH stress response mediates killing of Mycobacterium tuberculosis by activating nitronaphthofuran prodrugs via induction of Mrx2 expression.

The emergence of drug-resistant Mycobacterium tuberculosis strains highlights the need to discover anti-tuberculosis drugs with novel mechanisms of action. Here we discovered a mycobactericidal strategy based on the prodrug activation of selected chemical derivatives classified as nitronaphthofurans (nNFs) mediated by the coordinated action of the sigH and mrx2 genes. The transcription factor SigH is a key regulator of an extensive transcriptional network that responds to oxidative, nitrosative, and heat stresses in M. tuberculosis. The nNF action induced the SigH stress response which in turn induced the mrx2 overexpression. The nitroreductase Mrx2 was found to activate nNF prodrugs, killing replicating, non-replicating and intracellular forms of M. tuberculosis. Analysis of SigH DNA sequences obtained from spontaneous nNF-resistant M. tuberculosis mutants suggests disruption of SigH binding to the mrx2 promoter site and/or RNA polymerase core, likely promoting the observed loss of transcriptional control over Mrx2. Mutations found in mrx2 lead to structural defects in the thioredoxin fold of the Mrx2 protein, significantly impairing the activity of the Mrx2 enzyme against nNFs. Altogether, our work brings out the SigH/Mrx2 stress response pathway as a promising target for future drug discovery programs.

Trapping and retaining intermediates in glycosyltransferases.

Glycosyltransferases (GTs) attach sugar molecules to a broad range of acceptors, generating a remarkable amount of structural diversity in biological systems. GTs are classified as either "retaining" or "inverting" enzymes. Most retaining GTs typically use an SNi mechanism. In a recent article in the JBC, Doyle et al. demonstrate a covalent intermediate in the dual-module KpsC GT (GT107) supporting a double displacement mechanism.

GH18 endo-ß-N-acetylglucosaminidases use distinct mechanisms to process hybrid-type N-linked glycans.

N-glycosylation is one of the most abundant posttranslational modifications of proteins, essential for many physiological processes, including protein folding, protein stability, oligomerization and aggregation, and molecular recognition events. Defects in the N-glycosylation pathway cause diseases that are classified as congenital disorders of glycosylation. The ability to manipulate protein N-glycosylation is critical not only to our fundamental understanding of biology but also for the development of new drugs for a wide range of human diseases. Chemoenzymatic synthesis using engineered endo-ß-N-acetylglucosaminidases (ENGases) has been used extensively to modulate the chemistry of N-glycosylated proteins. However, defining the molecular mechanisms by which ENGases specifically recognize and process N-glycans remains a major challenge. Here we present the X-ray crystal structure of the ENGase EndoBT-3987 from Bacteroides thetaiotaomicron in complex with a hybrid-type glycan product. In combination with alanine scanning mutagenesis, molecular docking calculations and enzymatic activity measurements conducted on a chemically engineered monoclonal antibody substrate unveil two mechanisms for hybrid-type recognition and processing by paradigmatic ENGases. Altogether, the experimental data provide pivotal insight into the molecular mechanism of substrate recognition and specificity for GH18 ENGases and further advance our understanding of chemoenzymatic synthesis and remodeling of homogeneous N-glycan glycoproteins.

The Phosphatidyl-myo-Inositol Dimannoside Acyltransferase PatA Is Essential for Mycobacterium tuberculosis Growth In Vitro and In Vivo.

Mycobacterium tuberculosis comprises an unusual cell envelope dominated by unique lipids and glycans that provides a permeability barrier against hydrophilic drugs and is central for its survival and virulence. Phosphatidyl-myo-inositol mannosides (PIMs) are glycolipids considered to be not only key structural components of the cell envelope but also the precursors of lipomannan (LM) and lipoarabinomannan (LAM), important lipoglycans implicated in host-pathogen interactions. Here, we focus on PatA, a membrane-associated acyltransferase that transfers a palmitoyl moiety from palmitoyl coenzyme A (palmitoyl-CoA) to the 6-position of the mannose ring linked to the 2-position of inositol in PIM1/PIM2 We validate that the function of PatA is vital for M. tuberculosisin vitro and in vivo We constructed a patA conditional mutant and showed that silencing patA is bactericidal in batch cultures. This phenotype was associated with significantly reduced levels of Ac1PIM2, an important structural component of the mycobacterial inner membrane. The requirement of PatA for viability was also demonstrated during macrophage infection and in a mouse model of infection, where a dramatic decrease in viable counts was observed upon silencing of the patA gene. This is reminiscent of the behavior of PimA, the mannosyltransferase that initiates the PIM pathway, also found to be essential for M. tuberculosis growth in vitro and in vivo Altogether, the experimental data highlight the significance of the early steps of the PIM biosynthetic pathway for M. tuberculosis physiology and reveal that PatA is a novel target for drug discovery programs against this major human pathogen.IMPORTANCE Tuberculosis (TB) is the leading cause of death from a single infectious agent. The emergence of drug resistance in strains of M. tuberculosis, the etiologic agent of TB, emphasizes the need to identify new targets and antimicrobial agents. The mycobacterial cell envelope is a major factor in this intrinsic drug resistance. Here, we have focused on the biosynthesis of PIMs, key virulence factors and important components of the cell envelope. Specifically, we have determined that PatA, the acyltransferase responsible for the first acylation step of the PIM synthesis pathway, is essential in M. tuberculosis These results highlight the importance of early steps of the PIM biosynthetic pathway for mycobacterial physiology and the suitability of PatA as a potential new drug target.

Unveiling the activation dynamics of a fold-switch bacterial glycosyltransferase by 19F NMR.

Fold-switch pathways remodel the secondary structure topology of proteins in response to the cellular environment. It is a major challenge to understand the dynamics of these folding processes. Here, we conducted an in-depth analysis of the α-helix-to-ß-strand and ß-strand-to-α-helix transitions and domain motions displayed by the essential mannosyltransferase PimA from mycobacteria. Using 19F NMR, we identified four functionally relevant states of PimA that coexist in dynamic equilibria on millisecond-to-second timescales in solution. We discovered that fold-switching is a slow process, on the order of seconds, whereas domain motions occur simultaneously but are substantially faster, on the order of milliseconds. Strikingly, the addition of substrate accelerated the fold-switching dynamics of PimA. We propose a model in which the fold-switching dynamics constitute a mechanism for PimA activation.

Membrane fluidity adjusts the insertion of the transacylase PlsX to regulate phospholipid biosynthesis in Gram-positive bacteria.

PlsX plays a central role in the coordination of fatty acid and phospholipid biosynthesis in Gram-positive bacteria. PlsX is a peripheral membrane acyltransferase that catalyzes the conversion of acyl-ACP to acyl-phosphate, which is in turn utilized by the polytopic membrane acyltransferase PlsY on the pathway of bacterial phospholipid biosynthesis. We have recently studied the interaction between PlsX and membrane phospholipids in vivo and in vitro, and observed that membrane association is necessary for the efficient transfer of acyl-phosphate to PlsY. However, understanding the molecular basis of such a channeling mechanism remains a major challenge. Here, we disentangle the binding and insertion events of the enzyme to the membrane, and the subsequent catalysis. We show that PlsX membrane binding is a process mostly mediated by phospholipid charge, whereas fatty acid saturation and membrane fluidity remarkably influence the membrane insertion step. Strikingly, the PlsXL254E mutant, whose biological functionality was severely compromised in vivo but remains catalytically active in vitro, was able to superficially bind to phospholipid vesicles, nevertheless, it loses the insertion capacity, strongly supporting the importance of membrane insertion in acyl-phosphate delivery. We propose a mechanism in which membrane fluidity governs the insertion of PlsX and thus regulates the biosynthesis of phospholipids in Gram-positive bacteria. This model may be operational in other peripheral membrane proteins with an unprecedented impact in drug discovery/development strategies.

Conformational entropy of a single peptide controlled under force governs protease recognition and catalysis.

An immense repertoire of protein chemical modifications catalyzed by enzymes is available as proteomics data. Quantifying the impact of the conformational dynamics of the modified peptide remains challenging to understand the decisive kinetics and amino acid sequence specificity of these enzymatic reactions in vivo, because the target peptide must be disordered to accommodate the specific enzyme-binding site. Here, we were able to control the conformation of a single-molecule peptide chain by applying mechanical force to activate and monitor its specific cleavage by a model protease. We found that the conformational entropy impacts the reaction in two distinct ways. First, the flexibility and accessibility of the substrate peptide greatly increase upon mechanical unfolding. Second, the conformational sampling of the disordered peptide drives the specific recognition, revealing force-dependent reaction kinetics. These results support a mechanism of peptide recognition based on conformational selection from an ensemble that we were able to quantify with a torsional free-energy model. Our approach can be used to predict how entropy affects site-specific modifications of proteins and prompts conformational and mechanical selectivity.

Dissecting the Structural and Chemical Determinants of the "Open-to-Closed" Motion in the Mannosyltransferase PimA from Mycobacteria.

The phosphatidyl-myo-inositol mannosyltransferase A (PimA) is an essential peripheral membrane glycosyltransferase that initiates the biosynthetic pathway of phosphatidyl-myo-inositol mannosides (PIMs), key structural elements and virulence factors of Mycobacterium tuberculosis. PimA undergoes functionally important conformational changes, including (i) α-helix-to-ß-strand and ß-strand-to-α-helix transitions and (ii) an "open-to-closed" motion between the two Rossmann-fold domains, a conformational change that is necessary to generate a catalytically competent active site. In previous work, we established that GDP-Man and GDP stabilize the enzyme and facilitate the switch to a more compact active state. To determine the structural contribution of the mannose ring in such an activation mechanism, we analyzed a series of chemical derivatives, including mannose phosphate (Man-P) and mannose pyrophosphate-ribose (Man-PP-RIB), and additional GDP derivatives, such as pyrophosphate ribose (PP-RIB) and GMP, by the combined use of X-ray crystallography, limited proteolysis, circular dichroism, isothermal titration calorimetry, and small angle X-ray scattering methods. Although the ß-phosphate is present, we found that the mannose ring, covalently attached to neither phosphate (Man-P) nor PP-RIB (Man-PP-RIB), does promote the switch to the active compact form of the enzyme. Therefore, the nucleotide moiety of GDP-Man, and not the sugar ring, facilitates the "open-to-closed" motion, with the ß-phosphate group providing the high-affinity binding to PimA. Altogether, the experimental data contribute to a better understanding of the structural determinants involved in the "open-to-closed" motion not only observed in PimA but also visualized and/or predicted in other glycosyltransfeases. In addition, the experimental data might prove to be useful for the discovery and/or development of PimA and/or glycosyltransferase inhibitors.

Structure-function relationships underlying the dual N-acetylmuramic and N-acetylglucosamine specificities of the bacterial peptidoglycan deacetylase PdaC.

Bacillus subtilis PdaC (BsPdaC) is a membrane-bound, multidomain peptidoglycan N-deacetylase acting on N-acetylmuramic acid (MurNAc) residues and conferring lysozyme resistance to modified cell wall peptidoglycans. BsPdaC contains a C-terminal family 4 carbohydrate esterase (CE4) catalytic domain, but unlike other MurNAc deacetylases, BsPdaC also has GlcNAc deacetylase activity on chitooligosaccharides (COSs), characteristic of chitin deacetylases. To uncover the molecular basis of this dual activity, here we determined the X-ray structure of the BsPdaC CE4 domain at 1.54 Å resolution and analyzed its mode of action on COS substrates. We found that the minimal substrate is GlcNAc3 and that activity increases with the degree of glycan polymerization. COS deacetylation kinetics revealed that BsPdaC operates by a multiple-chain mechanism starting at the internal GlcNAc units and leading to deacetylation of all but the reducing-end GlcNAc residues. Interestingly, BsPdaC shares higher sequence similarity with the peptidoglycan GlcNAc deacetylase SpPgdaA than with other MurNAc deacetylases. Therefore, we used ligand-docking simulations to analyze the dual GlcNAc- and MurNAc-binding specificities of BsPdaC and compared them with those of SpPgdA and BsPdaA, representing peptidoglycan deacetylases highly specific for GlcNAc or MurNAc residues, respectively. BsPdaC retains the conserved Asp-His-His metal-binding triad characteristic of CE4 enzymes acting on GlcNAc residues, differing from MurNAc deacetylases that lack the metal-coordinating Asp residue. BsPdaC contains short loops similar to those in SpPgdA, resulting in an open binding cleft that can accommodate polymeric substrates. We propose that PdaC is the first member of a new subclass of peptidoglycan MurNAc deacetylases.

Structural insights into the mechanisms and specificities of IgG-active endoglycosidases.

The conserved N-glycan on Asn297 of immunoglobulin G (IgG) has significant impacts on antibody effector functions, and is a frequent target for antibody engineering. Chemoenzymatic synthesis has emerged as a strategy for producing antibodies with homogenous glycosylation and improved effector functions. Central to this strategy is the use of enzymes with activity on the Asn297 glycan. EndoS and EndoS2, produced by Streptococcus pyogenes, are endoglycosidases with remarkable specificity for Asn297 glycosylation, making them ideal tools for chemoenzymatic synthesis. Although both enzymes are specific for IgG, EndoS2 recognizes a wider range of glycans than EndoS. Recent progress has been made in understanding the structural basis for their activities on antibodies. In this review, we examine the molecular mechanism of glycosidic bond cleavage by these enzymes and how specific point mutations convert them into glycosynthases. We also discuss the structural basis for differences in the glycan repertoire that IgG-active endoglycosidases recognize, which focuses on the structure of the loops within the glycoside hydrolase (GH) domain. Finally, we discuss the important contributions of carbohydrate binding modules (CBMs) to endoglycosidase activity, and how CBMs work in concert with GH domains to produce optimal activity on IgG.

Structural basis of glycogen metabolism in bacteria.

The evolution of metabolic pathways is a major force behind natural selection. In the spotlight of such process lies the structural evolution of the enzymatic machinery responsible for the central energy metabolism. Specifically, glycogen metabolism has emerged to allow organisms to save available environmental surplus of carbon and energy, using dedicated glucose polymers as a storage compartment that can be mobilized at future demand. The origins of such adaptive advantage rely on the acquisition of an enzymatic system for the biosynthesis and degradation of glycogen, along with mechanisms to balance the assembly and disassembly rate of this polysaccharide, in order to store and recover glucose according to cell energy needs. The first step in the classical bacterial glycogen biosynthetic pathway is carried out by the adenosine 5'-diphosphate (ADP)-glucose pyrophosphorylase. This allosteric enzyme synthesizes ADP-glucose and acts as a point of regulation. The second step is carried out by the glycogen synthase, an enzyme that generates linear α-(1→4)-linked glucose chains, whereas the third step catalyzed by the branching enzyme produces α-(1→6)-linked glucan branches in the polymer. Two enzymes facilitate glycogen degradation: glycogen phosphorylase, which functions as an α-(1→4)-depolymerizing enzyme, and the debranching enzyme that catalyzes the removal of α-(1→6)-linked ramifications. In this work, we rationalize the structural basis of glycogen metabolism in bacteria to the light of the current knowledge. We describe and discuss the remarkable progress made in the understanding of the molecular mechanisms of substrate recognition and product release, allosteric regulation and catalysis of all those enzymes.

The Mycobacterium tuberculosis capsule: a cell structure with key implications in pathogenesis.

Bacterial capsules have evolved to be at the forefront of the cell envelope, making them an essential element of bacterial biology. Efforts to understand the Mycobacterium tuberculosis (Mtb) capsule began more than 60 years ago, but the relatively recent development of mycobacterial genetics combined with improved chemical and immunological tools have revealed a more refined view of capsule molecular composition. A glycogen-like α-glucan is the major constituent of the capsule, with lower amounts of arabinomannan and mannan, proteins and lipids. The major Mtb capsular components mediate interactions with phagocytes that favor bacterial survival. Vaccination approaches targeting the mycobacterial capsule have proven successful in controlling bacterial replication. Although the Mtb capsule is composed of polysaccharides of relatively low complexity, the concept of antigenic variability associated with this structure has been suggested by some studies. Understanding how Mtb shapes its envelope during its life cycle is key to developing anti-infective strategies targeting this structure at the host-pathogen interface.

3-Phosphoglycerate Transhydrogenation Instead of Dehydrogenation Alleviates the Redox State Dependency of Yeast de Novo l-Serine Synthesis.

The enzymatic mechanism of 3-phosphoglycerate to 3-phosphohydroxypyruvate oxidation, which forms the first step of the main conserved de novo serine synthesis pathway, has been revisited recently in certain microorganisms. While this step is classically considered to be catalyzed by an NAD-dependent dehydrogenase (e.g., PHGDH in mammals), evidence has shown that in Pseudomonas, Escherichia coli, and Saccharomyces cerevisiae, the PHGDH homologues act as transhydrogenases. As such, they use α-ketoglutarate, rather than NAD+, as the final electron acceptor, thereby producing D-2-hydroxyglutarate in addition to 3-phosphohydroxypyruvate during 3-phosphoglycerate oxidation. Here, we provide a detailed biochemical and sequence-structure relationship characterization of the yeast PHGDH homologues, encoded by the paralogous SER3 and SER33 genes, in comparison to the human and other PHGDH enzymes. Using in vitro assays with purified recombinant enzymes as well as in vivo growth phenotyping and metabolome analyses of yeast strains engineered to depend on either Ser3, Ser33, or human PHGDH for serine synthesis, we confirmed that both yeast enzymes act as transhydrogenases, while the human enzyme is a dehydrogenase. In addition, we show that the yeast paralogs differ from the human enzyme in their sensitivity to inhibition by serine as well as hydrated NADH derivatives. Importantly, our in vivo data support the idea that a 3PGA transhydrogenase instead of dehydrogenase activity confers a growth advantage under conditions where the NAD+:NADH ratio is low. The results will help to elucidate why different species evolved different reaction mechanisms to carry out a widely conserved metabolic step in central carbon metabolism.

Mechanistic insights into the allosteric regulation of bacterial ADP-glucose pyrophosphorylases.

ADP-glucose pyrophosphorylase (AGPase) controls bacterial glycogen and plant starch biosynthetic pathways, the most common carbon storage polysaccharides in nature. AGPase activity is allosterically regulated by a series of metabolites in the energetic flux within the cell. Very recently, we reported the first crystal structures of the paradigmatic AGPase from Escherichia coli (EcAGPase) in complex with its preferred physiological negative and positive allosteric regulators, adenosine 5'-monophosphate (AMP) and fructose 1,6-bisphosphate (FBP), respectively. However, understanding the molecular mechanism by which AMP and FBP allosterically modulates EcAGPase enzymatic activity still remains enigmatic. Here we found that single point mutations of key residues in the AMP-binding site decrease its inhibitory effect but also clearly abolish the overall AMP-mediated stabilization effect in wild-type EcAGPase. Single point mutations of key residues for FBP binding did not revert the AMP-mediated stabilization. Strikingly, an EcAGPase-R130A mutant displayed a dramatic increase in activity when compared with wild-type EcAGPase, and this increase correlated with a significant increment of glycogen content in vivo The crystal structure of EcAGPase-R130A revealed unprecedented conformational changes in structural elements involved in the allosteric signal transmission. Altogether, we propose a model in which the positive and negative energy reporters regulate AGPase catalytic activity via intra- and interprotomer cross-talk, with a "sensory motif" and two loops, RL1 and RL2, flanking the ATP-binding site playing a significant role. The information reported herein provides exciting possibilities for industrial/biotechnological applications.

The antibacterial prodrug activator Rv2466c is a mycothiol-dependent reductase in the oxidative stress response of Mycobacterium tuberculosis.

The Mycobacterium tuberculosis rv2466c gene encodes an oxidoreductase enzyme annotated as DsbA. It has a CPWC active-site motif embedded within its thioredoxin fold domain and mediates the activation of the prodrug TP053, a thienopyrimidine derivative that kills both replicating and nonreplicating bacilli. However, its mode of action and actual enzymatic function in M. tuberculosis have remained enigmatic. In this study, we report that Rv2466c is essential for bacterial survival under H2O2 stress. Further, we discovered that Rv2466c lacks oxidase activity; rather, it receives electrons through the mycothiol/mycothione reductase/NADPH pathway to activate TP053, preferentially via a dithiol-disulfide mechanism. We also found that Rv2466c uses a monothiol-disulfide exchange mechanism to reduce S-mycothiolated mixed disulfides and intramolecular disulfides. Genetic, phylogenetic, bioinformatics, structural, and biochemical analyses revealed that Rv2466c is a novel mycothiol-dependent reductase, which represents a mycoredoxin cluster of enzymes within the DsbA family different from the glutaredoxin cluster to which mycoredoxin-1 (Mrx1 or Rv3198A) belongs. To validate this DsbA-mycoredoxin cluster, we also characterized a homologous enzyme of Corynebacterium glutamicum (NCgl2339) and observed that it demycothiolates and reduces a mycothiol arsenate adduct with kinetic properties different from those of Mrx1. In conclusion, our work has uncovered a DsbA-like mycoredoxin that promotes mycobacterial resistance to oxidative stress and reacts with free mycothiol and mycothiolated targets. The characterization of the DsbA-like mycoredoxin cluster reported here now paves the way for correctly classifying similar enzymes from other organisms.

Molecular Basis of Membrane Association by the Phosphatidylinositol Mannosyltransferase PimA Enzyme from Mycobacteria.

Phosphatidyl-myo-inositol mannosyltransferase A (PimA) is an essential glycosyltransferase that initiates the biosynthetic pathway of phosphatidyl-myo-inositol mannoside, lipomannan, and lipoarabinomannan, which are key glycolipids/lipoglycans of the mycobacterial cell envelope. PimA belongs to a large family of membrane-associated glycosyltransferases for which the understanding of the molecular mechanism and conformational changes that govern substrate/membrane recognition and catalysis remains a major challenge. Here, we determined that PimA preferentially binds to negatively charged phosphatidyl-myo-inositol substrate and non-substrate membrane model systems (small unilamellar vesicle) through its N-terminal domain, inducing an important structural reorganization of anionic phospholipids. By using a combination of single-point mutagenesis, circular dichroism, and a variety of fluorescence spectroscopy techniques, we determined that this interaction is mainly mediated by an amphipathic α-helix (α2), which undergoes a substantial conformational change and localizes in the vicinity of the negatively charged lipid headgroups and the very first carbon atoms of the acyl chains, at the PimA-phospholipid interface. Interestingly, a flexible region within the N-terminal domain, which undergoes ß-strand-to-α-helix and α-helix-to-ß-strand transitions during catalysis, interacts with anionic phospholipids; however, the effect is markedly less pronounced to that observed for the amphipathic α2, likely reflecting structural plasticity/variability. Altogether, we propose a model in which conformational transitions observed in PimA might reflect a molten globule state that confers to PimA, a higher affinity toward the dynamic and highly fluctuating lipid bilayer.