Menaquinone Biosynthesis Research Articles

Site-directed mutagenesis of UbiA to promote menaquinone biosynthesis in Elizabethkingia meningoseptica

Abstract To increase the menaquinone (MK) content of an Elizabethkingia meningoseptica, site-directed mutagenesis was generated to suppress 4-hydroxybenzoate octaprenyl transferase (UbiA) activity and subsequently blocked the ubiquinone (UQ) biosynthesis pathway. Fourteen conserved residues except L174 and G211 were mutated to analyze the effect of site-directed mutagenesis. The expression of UbiA in twelve mutants was decreased in both mRNA and protein levels, which resulted in the decrease of UQ concentration. Based on MenA expression level, 12 mutants were divided into two groups. Second group such as N72A, D76A, K81A, L139A, and D198A enhanced the expression of MenA, which increased MK production by 127.1%, 87.9%, 96.2%, 109.7% and 130.0% in wt-EmUbiA, respectively. In general, blocking UQ synthesis pathway for by site-directed mutagenesis of the active site of UbiA in E. meningoseptica was a promising strategy to increase MK production in E. meningoseptica.

A Novel Small-Molecule Inhibitor of the Mycobacterium tuberculosis Demethylmenaquinone Methyltransferase MenG Is Bactericidal to Both Growing and Nutritionally Deprived Persister Cells.

ABSTRACTActive tuberculosis (TB) and latent Mycobacterium tuberculosis infection both require lengthy treatments to achieve durable cures. This problem has partly been attributable to the existence of nonreplicating M. tuberculosis “persisters” that are difficult to kill using conventional anti-TB treatments. Compounds that target the respiratory pathway have the potential to kill both replicating and persistent M. tuberculosis and shorten TB treatment, as this pathway is essential in both metabolic states. We developed a novel respiratory pathway-specific whole-cell screen to identify new respiration inhibitors. This screen identified the biphenyl amide GSK1733953A (DG70) as a likely respiration inhibitor. DG70 inhibited both clinical drug-susceptible and drug-resistant M. tuberculosis strains. Whole-genome sequencing of DG70-resistant colonies identified mutations in menG (rv0558), which is responsible for the final step in menaquinone biosynthesis and required for respiration. Overexpression of menG from wild-type and DG70-resistant isolates increased the DG70 MIC by 4× and 8× to 30×, respectively. Radiolabeling and high-resolution mass spectrometry studies confirmed that DG70 inhibited the final step in menaquinone biosynthesis. DG70 also inhibited oxygen utilization and ATP biosynthesis, which was reversed by external menaquinone supplementation. DG70 was bactericidal in actively replicating cultures and in a nutritionally deprived persistence model. DG70 was synergistic with the first-line TB drugs isoniazid, rifampin, and the respiratory inhibitor bedaquiline. The combination of DG70 and isoniazid completely sterilized cultures in the persistence model by day 10. These results suggest that MenG is a good therapeutic target and that compounds targeting MenG along with standard TB therapy have the potential to shorten TB treatment duration.

Novel long-chain compounds with both immunomodulatory and MenA inhibitory activities against Staphylococcus aureus and its biofilm

Menaquinone (MK) biosynthesis pathway is a potential target for evaluating antimicrobials in gram-positive bacteria. Here, 1,4-dihydroxy-2-naphthoate prenyltransferase (MenA) was targeted to reduce methicillin-resistant Staphylococcus aureus (MRSA) growth. MenA inhibiting, long chain-based compounds were designed, synthesized and evaluated against MRSA and menaquinone utilizing bacteria in aerobic conditions. The results showed that these bacteria were susceptible to most of the compounds. Menaquinone (MK-4) supplementation rescued MRSA growth, suggesting these compounds inhibit MK biosynthesis. 3a and 7c exhibited promising inhibitory activities with MICs ranging 1–8 μg/mL against MRSA strains. The compounds did not facilitate small colony variant formation. These compounds also inhibited the biofilm growth by MRSA at high concentration. Compounds 3a, 6b and 7c displayed a promising extracellular bactericidal activity against MRSA at concentrations equal to and four-fold less than their respective MICs. We also observed cytokines released from THP-1 macrophages treated with compounds 3a, 6b and 7c and found decreases in TNF-α and IL-6 release and increase in IL-1β. These data provide evidence that MenA inhibitors act as TNF-α and IL-6 inhibitors, raising the potential for development and application of these compounds as potential immunomodulatory agents.

Stereoselective Synthesis, Docking, and Biological Evaluation of Difluoroindanediol-Based MenE Inhibitors as Antibiotics.

A stereoselective synthesis has been developed to provide all four side-chain stereoisomers of difluoroindanediol 2, the mixture of which was previously identified as an inhibitor of the o-succinylbenzoate-CoA synthetase MenE in bacterial menaquinone biosynthesis, having promising in vitro activity against methicillin-resistant Staphylococcus aureus and Mycobacterium tuberculosis. Only the (1R,3S)-diastereomer inhibited the biochemical activity of MenE, consistent with computational docking studies, and this diastereomer also exhibited in vitro antibacterial activity comparable to that of the mixture. However, mechanism-of-action studies suggest that this inhibitor and its diastereomers may act via other mechanisms beyond inhibition of menaquinone biosynthesis.

Novel Pathways for Ameliorating the Fitness Cost of Gentamicin Resistant Small Colony Variants.

Small colony variants (SCVs) of the human pathogen Staphylococcus aureus are associated with persistent infections. Phenotypically, SCVs are characterized by slow growth and they can arise upon interruption of the electron transport chain that consequently reduce membrane potential and thereby limit uptake of aminoglycosides (e.g., gentamicin). In this study, we have examined the pathways by which the fitness cost of SCVs can be ameliorated. Five gentamicin resistant SCVs derived from S. aureus JE2 were independently selected on agar plates supplemented with gentamicin. The SCVs carried mutations in the menaquinone and hemin biosynthesis pathways, which caused a significant reduction in exponential growth rates relative to wild type (WT; 0.59–0.72) and reduced membrane potentials. Fifty independent lineages of the low-fitness, resistant mutants were serially passaged for up to 500 generations with or without sub-lethal concentrations of gentamicin. Amelioration of the fitness cost followed three evolutionary trajectories and was dependent on the initial mutation type (point mutation vs. deletion) and the passage condition (absence or presence of gentamicin). For SCVs evolved in the absence of gentamicin, 12 out of 15 lineages derived from SCVs with point mutations acquired intra-codonic suppressor mutations restoring membrane potential, growth rate, gentamicin susceptibility and colony size to WT levels. For the SCVs carrying deletions, all lineages enhanced fitness independent of membrane potential restoration without alterations in gentamicin resistance levels. By whole genome sequencing, we identified compensatory mutations in genes related to the σB stress response (7 out of 10 lineages). Inactivation of rpoF that encode for the alternative sigma factor SigB (σB) partially restored fitness of SCVs. For all lineages passaged in the presence of gentamicin, fitness compensation via membrane potential restoration was suppressed, however, selected for secondary mutations in fusA and SAUSA300_0749. This study is the first to describe fitness compensatory events in SCVs with deletion mutations and adaptation of SCVs to continued exposure to gentamicin.

Structure, Function, and Inhibition of Staphylococcus aureus Heptaprenyl Diphosphate Synthase.

We report the first structure of heptaprenyl diphosphate synthase from Staphylococcus aureus (SaHepPPS), together with an investigation of its mechanism of action and inhibition. The protein is involved in the formation of menaquinone, a key electron transporter in many bacteria, including pathogens. SaHepPPS consists of a "catalytic " subunit (SaHepPPS-2) having two "DDXXD" motifs and a "regulatory" subunit (SaHepPPS-1) that lacks these motifs. High concentrations of the substrates, isopentenyl diphosphate and farnesyl diphosphate, inhibit the enzyme, which is also potently inhibited by bisphosphonates. The most active inhibitors (Ki ∼200 nm) were N-alkyl analogues of zoledronate containing ∼C6 alkyl side chains. They were modestly active against S. aureus cell growth, and growth inhibition was partially "rescued" by the addition of menaquinone-7. Because SaHepPPS is essential for S. aureus cell growth, its structure is of interest in the context of the development of menaquinone biosynthesis inhibitors as potential antibiotic leads.

Structural Views along the Mycobacterium tuberculosis MenD Reaction Pathway Illuminate Key Aspects of Thiamin Diphosphate-Dependent Enzyme Mechanisms

Menaquinone (MQ) is an essential component of the respiratory chains of many pathogenic organisms, including Mycobacterium tuberculosis (Mtb). The first committed step in MQ biosynthesis is catalyzed by 2-succinyl-5-enolpyruvyl-6-hydroxy-3-cyclohexadiene-1-carboxylate synthase (MenD), a thiamin diphosphate (ThDP)-dependent enzyme. Catalysis proceeds through two covalent intermediates as the substrates 2-oxoglutarate and isochorismate are successively added to the cofactor before final cleavage of the product. We have determined a series of crystal structures of Mtb-MenD that map the binding of both substrates, visualizing each step in the MenD catalytic cycle, including both intermediates. ThDP binding induces a marked asymmetry between the coupled active sites of each dimer, and possible mechanisms of communication can be identified. The crystal structures also reveal conformational features of the two intermediates that facilitate reaction but prevent premature product release. These data fully map chemical space to inform early-stage drug discovery targeting MenD.

Menaquinone Biosynthesis: An Antibacterial Target?

Menaquinone Biosynthesis: An Antibacterial Target?

A Methyl 4-Oxo-4-phenylbut-2-enoate with in Vivo Activity against MRSA that Inhibits MenB in the Bacterial Menaquinone Biosynthesis Pathway.

4-Oxo-4-phenyl-but-2-enoates inhibit MenB, the 1,4-dihydroxyl-2-naphthoyl-CoA synthase in the bacterial menaquinone (MK) biosynthesis pathway, through the formation of an adduct with coenzyme A (CoA). Here, we show that the corresponding methyl butenoates have MIC values as low as 0.35-0.75 µg/mL against drug sensitive and resistant strains of Staphylococcus aureus. Mode of action studies on the most potent compound, methyl 4-(4-chlorophenyl)-4-oxobut-2-enoate (1), reveal that 1 is converted into the corresponding CoA adduct in S. aureus cells, and that this adduct binds to the S. aureus MenB (saMenB) with a Kd value of 2 µM. The antibacterial spectrum of 1 is limited to bacteria that utilize MK for respiration, and the activity of 1 can be complemented with exogenous MK or menadione. Finally, treatment of methicillin-resistant S. aureus (MRSA) with 1 results in the small colony variant phenotype and thus 1 phenocopies knockout of the menB gene. Taken together the data indicate that the antibacterial activity of 1 results from a specific effect on MK biosynthesis. We also evaluated the in vivo efficacy of 1 using two mouse models of MRSA infection. Notably, compound 1 increased survival in a systemic infection model and resulted in a dose-dependent decrease in bacterial load in a thigh infection model, validating MenB as a target for the development of new anti-MRSA candidates.

Characterization and engineering of the biosynthesis gene cluster for antitumor macrolides PM100117 and PM100118 from a marine actinobacteria: generation of a novel improved derivative.

BackgroundPM100117 and PM100118 are glycosylated polyketides with remarkable antitumor activity, which derive from the marine symbiotic actinobacteria Streptomyces caniferus GUA-06-05-006A. Structurally, PM100117 and PM100118 are composed of a macrocyclic lactone, three deoxysugar units and a naphthoquinone (NQ) chromophore that shows a clear structural similarity to menaquinone.ResultsWhole-genome sequencing of S. caniferus GUA-06-05-006A has enabled the identification of PM100117 and PM100118 biosynthesis gene cluster, which has been characterized on the basis of bioinformatics and genetic engineering data. The product of four genes shows high identity to proteins involved in the biosynthesis of menaquinone via futalosine. Deletion of one of these genes led to a decay in PM100117 and PM100118 production, and to the accumulation of several derivatives lacking NQ. Likewise, five additional genes have been genetically characterized to be involved in the biosynthesis of this moiety. Moreover, the generation of a mutant in a gene coding for a putative cytochrome P450 has led to the production of PM100117 and PM100118 structural analogues showing an enhanced in vitro cytotoxic activity relative to the parental products.ConclusionsAlthough a number of compounds structurally related to PM100117 and PM100118 has been discovered, this is, to our knowledge, the first insight reported into their biosynthesis. The structural resemblance of the NQ moiety to menaquinone, and the presence in the cluster of four putative menaquinone biosynthetic genes, suggests a connection between the biosynthesis pathways of both compounds. The availability of the PM100117 and PM100118 biosynthetic gene cluster will surely pave a way to the combinatorial engineering of more derivatives.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-016-0443-5) contains supplementary material, which is available to authorized users.

Menaquinone as a potential target of antibacterial agents.

The current trend of increasing infections by multidrug-resistant pathogens requires the discovery of novel antimicrobial agents with new target and selective toxicity towards pathogens. Menaquinone is a component of electron transport chains in a majority of anaerobic bacteria and Gram-positive bacteria. Due to its exclusivity in bacteria, menaquinone is thought to be a potential target for development of therapeutically effective antibacterial agents without side effects. In this review, we summarize inhibitors of menaquinone biosynthesis and antibiotics directly targeting menaquinone in bacteria.

Discovery of bicyclic inhibitors against menaquinone biosynthesis.

Menaquinone is used for transporting electrons and is essential for the aerobic and anaerobic respiratory systems of all pathogens and prokaryotes. Many Gram-positive bacteria use only menaquinone in the electron transport system. Thus, menaquinone biosynthesis is a potential target for the development of inhibitors against bacteria including drug-resistant pathogens. After modeling, synthesis and in vitro testing, we determined that 7-methoxy-2-naphthol-based inhibitors targeted the MenA enzyme of the menaquinone biosynthesis pathway. The developmental compounds 1 and 2 were active against Mycobacterium tuberculosis and methicillin-resistant Staphylococcus aureus with a minimal inhibitory concentration of 3-5 μg/ml. Nontraditional bicyclic inhibitors, compounds 1 and 2 could serve as lead compounds for the development of an antimicrobial agent, with activities against M. tuberculosis and methicillin-resistant S. aureus.

New Antibiotic Candidates against Helicobacter pylori.

Helicobacter pylori is a Gram-negative bacterium that colonizes the gut of over 50% of the world's population. It is responsible for most peptic ulcers and is an important risk factor for gastric cancer. Antibiotic treatment for H. pylori infections is challenging as drug resistance has developed to antibiotics with traditional mechanisms of action. H. pylori uses an unusual pathway for menaquinone biosynthesis with 5'-methylthioadenosine/S-adenosylhomocysteine nucleosidase (MTAN) catalyzing an essential step. We validated MTAN as a target with a transition-state analogue of the enzyme [Wang, S.; Haapalainen, A. M.; Yan, F.; et al. Biochemistry 2012, 51, 6892-6894]. MTAN inhibitors will only be useful drug candidates if they can both include tight binding to the MTAN target and have the ability to penetrate the complex cell membrane found in Gram-negative H. pylori. Here we explore structural scaffolds for MTAN inhibition and for growth inhibition of cultured H. pylori. Sixteen analogues reported here are transition-state analogues of H. pylori MTAN with dissociation constants of 50 pM or below. Ten of these prevent growth of the H. pylori with IC90 values below 0.01 μg/mL. These remarkable compounds meet the criteria for potent inhibition and cell penetration. As a consequence, 10 new H. pylori antibiotic candidates are identified, all of which prevent H. pylori growth at concentrations 16-2000-fold lower than the five antibiotics, amoxicillin, metronidazole, levofloxacin, tetracyclin, and clarithromycin, commonly used to treat H. pylori infections. X-ray crystal structures of MTAN cocrystallized with several inhibitors show them to bind in the active site making interactions consistent with transition-state analogues.

Mechanism of MenE inhibition by acyl-adenylate analogues and discovery of novel antibacterial agents.

MenE is an o-succinylbenzoyl-CoA (OSB-CoA) synthetase in the bacterial menaquinone biosynthesis pathway and is a promising target for the development of novel antibacterial agents. The enzyme catalyzes CoA ligation via an acyl-adenylate intermediate, and we have previously reported tight-binding inhibitors of MenE based on stable acyl-sulfonyladenosine analogues of this intermediate, including OSB-AMS (1), which has an IC50 value of ≤25 nM for Escherichia coli MenE. Herein, we show that OSB-AMS reduces menaquinone levels in Staphylococcus aureus, consistent with its proposed mechanism of action, despite the observation that the antibacterial activity of OSB-AMS is ∼1000-fold lower than the IC50 for enzyme inhibition. To inform the synthesis of MenE inhibitors with improved antibacterial activity, we have undertaken a structure-activity relationship (SAR) study stimulated by the knowledge that OSB-AMS can adopt two isomeric forms in which the OSB side chain exists either as an open-chain keto acid or a cyclic lactol. These studies revealed that negatively charged analogues of the keto acid form bind, while neutral analogues do not, consistent with the hypothesis that the negatively charged keto acid form of OSB-AMS is the active isomer. X-ray crystallography and site-directed mutagenesis confirm the importance of a conserved arginine for binding the OSB carboxylate. Although most lactol isomers tested were inactive, a novel difluoroindanediol inhibitor (11) with improved antibacterial activity was discovered, providing a pathway toward the development of optimized MenE inhibitors in the future.

Structural Basis for the ATP-dependent Configuration of Adenylation Active Site in Bacillus subtilis o-Succinylbenzoyl-CoA Synthetase

o-Succinylbenzoyl-CoA synthetase, or MenE, is an essential adenylate-forming enzyme targeted for development of novel antibiotics in the menaquinone biosynthesis. Using its crystal structures in a ligand-free form or in complex with nucleotides, a conserved pattern is identified in the interaction between ATP and adenylating enzymes, including acyl/aryl-CoA synthetases, adenylation domains of nonribosomal peptide synthetases, and luciferases. It involves tight gripping interactions of the phosphate-binding loop (P-loop) with the ATP triphosphate moiety and an open-closed conformational change to form a compact adenylation active site. In MenE catalysis, this ATP-enzyme interaction creates a new binding site for the carboxylate substrate, allowing revelation of the determinants of substrate specificities and in-line alignment of the two substrates for backside nucleophilic substitution reaction by molecular modeling. In addition, the ATP-enzyme interaction is suggested to play a crucial catalytic role by mutation of the P-loop residues hydrogen-bonded to ATP. Moreover, the ATP-enzyme interaction has also clarified the positioning and catalytic role of a conserved lysine residue in stabilization of the transition state. These findings provide new insights into the adenylation half-reaction in the domain alteration catalytic mechanism of the adenylate-forming enzymes.

Mutation of Rv2887, a marR-like gene, confers Mycobacterium tuberculosis resistance to an imidazopyridine-based agent.

Drug resistance is a major problem in Mycobacterium tuberculosis control, and it is critical to identify novel drug targets and new antimycobacterial compounds. We have previously identified an imidazo[1,2-a]pyridine-4-carbonitrile-based agent, MP-III-71, with strong activity against M. tuberculosis. In this study, we evaluated mechanisms of resistance to MP-III-71. We derived three independent M. tuberculosis mutants resistant to MP-III-71 and conducted whole-genome sequencing of these mutants. Loss-of-function mutations in Rv2887 were common to all three MP-III-71-resistant mutants, and we confirmed the role of Rv2887 as a gene required for MP-III-71 susceptibility using complementation. The Rv2887 protein was previously unannotated, but domain and homology analyses suggested it to be a transcriptional regulator in the MarR (multiple antibiotic resistance repressor) family, a group of proteins first identified in Escherichia coli to negatively regulate efflux pumps and other mechanisms of multidrug resistance. We found that two efflux pump inhibitors, verapamil and chlorpromazine, potentiate the action of MP-III-71 and that mutation of Rv2887 abrogates their activity. We also used transcriptome sequencing (RNA-seq) to identify genes which are differentially expressed in the presence and absence of a functional Rv2887 protein. We found that genes involved in benzoquinone and menaquinone biosynthesis were repressed by functional Rv2887. Thus, inactivating mutations of Rv2887, encoding a putative MarR-like transcriptional regulator, confer resistance to MP-III-71, an effective antimycobacterial compound that shows no cross-resistance to existing antituberculosis drugs. The mechanism of resistance of M. tuberculosis Rv2887 mutants may involve efflux pump upregulation and also drug methylation.

Essential roles of methionine and S-adenosylmethionine in the autarkic lifestyle of Mycobacterium tuberculosis

Multidrug resistance, strong side effects, and compliance problems in TB chemotherapy mandate new ways to kill Mycobacterium tuberculosis (Mtb). Here we show that deletion of the gene encoding homoserine transacetylase (metA) inactivates methionine and S-adenosylmethionine (SAM) biosynthesis in Mtb and renders this pathogen exquisitely sensitive to killing in immunocompetent or immunocompromised mice, leading to rapid clearance from host tissues. Mtb ΔmetA is unable to proliferate in primary human macrophages, and in vitro starvation leads to extraordinarily rapid killing with no appearance of suppressor mutants. Cell death of Mtb ΔmetA is faster than that of other auxotrophic mutants (i.e., tryptophan, pantothenate, leucine, biotin), suggesting a particularly potent mechanism of killing. Time-course metabolomics showed complete depletion of intracellular methionine and SAM. SAM depletion was consistent with a significant decrease in methylation at the DNA level (measured by single-molecule real-time sequencing) and with the induction of several essential methyltransferases involved in biotin and menaquinone biosynthesis, both of which are vital biological processes and validated targets of antimycobacterial drugs. Mtb ΔmetA could be partially rescued by biotin supplementation, confirming a multitarget cell death mechanism. The work presented here uncovers a previously unidentified vulnerability of Mtb-the incapacity to scavenge intermediates of SAM and methionine biosynthesis from the host. This vulnerability unveils an entirely new drug target space with the promise of rapid killing of the tubercle bacillus by a new mechanism of action.

Fecal menaquinone profiles of overweight adults are associated with gut microbiota composition during a gut microbiota–targeted dietary intervention

Emerging evidence supports novel roles for vitamin K in cardiometabolic health, some of which may be unique to the bacterially synthesized vitamin K forms known as menaquinones. However, factors influencing menaquinone biosynthesis by the gut microbiota and associations with cardiometabolic health have not been examined. The objective of this study was to identify associations between fecal menaquinone profiles, gut microbiota composition, and biomarkers of cardiometabolic health. The menaquinone profile and gut microbiota structure were periodically measured in fecal samples collected from 77 overweight Chinese adults who consumed a prescribed diet previously shown to alter gut microbiota composition and to improve cardiometabolic biomarkers. Covariance among menaquinones within individual fecal samples partitioned individuals into 2 distinct groups, herein introduced as menaquinotypes of the human gut. Menaquinotypes were characterized by differences in menaquinone (MK) 5 and MK9-MK13 and differences in the relative abundance of several operational taxonomic units (OTUs) delineated at the species level, predominantly within the genera Prevotella spp. and Bacteroides spp. Fecal MK4, MK6, and MK8 decreased during the intervention (P < 0.05); and longitudinal changes in the relative abundance of >100 OTUs were associated with altered fecal content of ≥1 individual menaquinone. The strongest and most consistent relations were between Prevotella spp. and MK5 and MK11-MK13, between Bacteroides spp. and MK9 and MK10, and between Escherichia/Shigella spp. and MK8. Neither individual menaquinones nor menaquinotypes were longitudinally associated with markers of glycemia, insulin resistance, or inflammation. These findings suggest that variability in fecal menaquinone content is predominantly determined by relatively few genera within the gut microbiota and that diet-mediated alterations in gut microbiota composition may provide a feasible approach for altering gut menaquinone content. This trial was registered at the Chinese Clinical Trials Registry as ChiCTR-TRC-09000353.

High-Resolution Mass Spectrometry Based Proteomic Analysis of the Response to Vancomycin-Induced Cell Wall Stress in Streptomyces coelicolor A3(2).

Understanding how bacteria survive periods of cell wall stress is of fundamental interest and can help generate ideas for improved antibacterial treatments. In this study we use tandem mass tagging to characterize the proteomic response of vancomycin resistant Streptomyces coelicolor to the exposure to sublethal levels of the antibiotic. A common set of 804 proteins were identified in triplicate experiments. Contrasting changes in the abundance of proteins closely associated with the cytoplasmic membrane with those taking place in the cytosol identified aspects of protein spatial localization that are associated with the response to vancomycin. Enzymes for peptidoglycan precursor, mycothiol, ectoine and menaquinone biosynthesis together with a multisubunit nitrate reductase were recruited to the membrane following vancomycin treatment. Many proteins with regulatory functions (including sensor protein kinases) also exhibited significant changes in abundance exclusively in the membrane-associated protein fraction. Several enzymes predicted to be involved in extracellular peptidoglycan crossbridge formation became significantly depleted from the membrane. A comparison with data previously acquired on the changes in gene transcription following vancomycin treatment identified a common high-confidence set of changes in gene expression. Generalized changes in protein abundance indicate roles for proteolysis, the pentose phosphate pathway and a reorganization of amino acid biosynthesis in the stress response.

Ligand-dependent active-site closure revealed in the crystal structure of Mycobacterium tuberculosis MenB complexed with product analogues.

1,4-Dihydroxy-2-naphthoyl coenzyme A (DHNA-CoA) synthase catalyzes an essential intramolecular Claisen condensation in menaquinone biosynthesis and is an important target for the development of new antibiotics. This enzyme in Mycobacterium tuberculosis is cofactor-free and is classified as a type II DHNA-CoA synthase, differing from type I enzymes, which rely on exogenous bicarbonate for catalysis. Its crystal structures in complex with product analogues have been determined at high resolution to reveal ligand-dependent structural changes, which include the ordering of a 27-residue active-site loop (amino acids 107-133) and the reorientation of the carboxy-terminal helix (amino acids 289-301) that forms part of the active site from the opposing subunit across the trimer-trimer interface. These structural changes result in closure of the active site to the bulk solution, which is likely to take place through an induced-fit mechanism, similar to that observed for type I DHNA-CoA synthases. These findings demonstrate that the ligand-dependent conformational changes are a conserved feature of all DHNA-CoA synthases, providing new insights into the catalytic mechanism of this essential tubercular enzyme.