Enzymes of Fatty Acid Synthesis

The growing challenge of drug resistance has intensified the search for new therapeutic targets against the virulent pathogen Mycobacterium tuberculosis (Mtb). The complex cell envelope of Mtb contains unique lipids, such as mycolic acids, which contribute to its survival under hostile conditions. While modern drugs like isoniazid inhibit mycolic acid biosynthesis through the fatty acid synthase II (FAS II) complex, alternative bypass pathways may facilitate the emergence of drug resistance. HtdX, a putative β-hydroxyacyl dehydratase gene conserved in the mycobacterial species, is hypothesized to play a role in these alternative fatty acid metabolism pathways. Although HtdX is expressed under nutrient-deficient conditions, its structural and functional characterization remains largely unexplored. This study presents the crystal structures of HtdX, revealing a MaoC-like dehydratase with a double hot-dog fold. Site-directed mutagenesis, enzyme kinetics, and fluorescence spectroscopy highlight the critical roles of the α2-β2 loop and the proline rich PP-loop in substrate specificity. The α2-β2 loop determines fatty acyl chain length specificity, while the PP-loop regulates the interaction between HtdX and the acyl carrier protein (AcpM). Computational predictions, complemented by molecular dynamics simulations and principal component analyses, establish that the N-terminal region of HtdX is essential for membrane binding. Overall, these findings offer insights into HtdX substrate specificity and provide theoretical understanding of its interaction with the membrane.

Vibrio cholerae, the causative pathogen of the life-threatening infection cholera, encodes two copies of b-ketoacyl-acyl carrier protein synthase III (vcFabH1 and vcFabH2). vcFabH1 and vcFabH2 are pathogenic proteins associated with fatty acid synthesis, lipid metabolism, and potential applications in biofuel production. Our biochemical assays characterize vcFabH1 as exhibiting specificity for acetyl-CoA and CoA thioesters with short acyl chains, similar to that observed for FabH homologs found in most gramnegative bacteria. vcFabH2 prefers medium chain-length acyl-CoA thioesters, particularly octanoyl-CoA, which is a pattern of specificity rarely seen in bacteria. Structural characterization of one vcFabH1 and six vcFabH2 structures determined in either apo form or in complex with acetyl-CoA/ octanoyl-CoA indicate that the substrate-binding pockets of vcFabH1 and vcFabH2 are of different sizes, accounting for variations in substrate chainlength specificity. An unusual and unique feature of vcFabH2 is its C-terminal fragment that interacts with both the substrate-entrance loop and the dimer interface of the enzyme. Our discovery of the pattern of substrate specificity of both vcFabH1 and vcFabH2 can potentially aid the development of novel antibacterial agents against V. cholerae. Additionally, the distinctive substrate preference of FabH2 in V. cholerae and related facultative anaerobes conceivably make it an attractive component of genetically engineered bacteria used for commercial biofuel production.

AMP-activated protein kinase is a multisubstrate protein kinase that, in liver, inactivates both acetyl-CoA carboxylase, the rate-limiting enzyme of fatty acid synthesis, and 3-hydroxy-3-methyl-glutaryl-CoA reductase, the rate-limiting enzyme of cholesterol synthesis. AICAR (5-amino 4-imidazolecarboxamide ribotide, ZMP) was found to stimulate up to 10-fold rat liver AMP-activated protein kinase, with a half-maximal effect at approximately 5 mM. In accordance with previous observations, addition to suspensions of isolated rat hepatocytes of 50-500 eM AlCAriboside, the nucleoside corresponding to ZMP, resulted in the accumulation of millimolar concentrations of the latter. This was accompanied by a dose-dependent inactivation of both acetyl-CoA carboxylase and 3-hydroxy-3-methylglutaryl-CoA reductase. Addition of 50-500 M AlCAriboside to hepatocyte suspensions incubated in the presence of various substrates, including glucose and lactate/pyruvate, caused a parallel inhibition of both fatty acid and cholesterol synthesis. With lactate/pyruvate (10/1 mM), half-maximal inhibition was obtained at approximately 100 scM, and near-complete inhibition at 500 sM AL-CAriboside. These findings open new perspectives for the simultaneous control of triglyceride and cholesterol synthesis by pharmacological stimulators of AMP-activated protein kinase.-Henin, N., Vincent, M.-F., Gruber, H. E., Van den Berghe, G. Inhibition of fatty acid and cholesterol synthesis by stimulation of AMP-activated protein kinase.

Stimulation of AMP-activated protein kinase (AMP-PK) by ZMP (5-amino-4-imidazolecarboxamide ribotide, AICAR), formed by adenosine kinase upon addition of AICAriboside to isolated rat hepatocytes, results in inhibition of fatty acid and cholesterol synthesis by inactivation of acetyl-CoA carboxylase and 3-hydroxy-3-methylglutaryl-CoA reductase, respectively (Henin et al. (1995) FASEB J. 9, 541-546). The effects of ZMP and other AMP analogues have now been compared with those of AMP on AMP-PK purified from rat liver. ZMP stimulated AMP-PK to the same maximal extent as AMP (about 10-fold). ZMP had less affinity for AMP-PK than AMP, but this affinity was similarly influenced by ATP: half-maximal effects, requiring 0.4 mM AMP or 5 mM ZMP at 3 mM ATP, were obtained with 9 txM AMP or 0.4 mM ZMP at 0.2 mM ATP. The kinetic parameters of AMP-PK for the SAMS peptide and for ATP were influenced in the same way by ZMP and AMP. Stimulation of AMP-PK by ZMP was additive with AMP, up to when maximal stimulation was obtained. Taken together, these results indicate that ZMP binds to the same site as AMP on AMP-PK. Tubercidin 5'-monophosphate, 2'-deoxy-AMP and Ara-AMP stimulated AMP-PK, but N6-methyl-AMP, 1,N6-etheno-AMP, 6-mercaptopurine riboside 5'-monophosphate, adenylosuccinate and succinyl-AICAR were ineffective, suggesting that a free 6-NH 2 group may be important for binding of effectors to AMP-PK.

Pathogenic bacteria resistant to most antibiotics, including the methicillin-resistant Staphylococcus aureus (MRSA) represent a serious medical problem. The search for new antiinfectives, possessing a diverse mechanism of action compared to the clinically used antibiotics, has become an attractive research field. S. aureus DNA encodes a b-class carbonic anhydrase, SauBCA. It is a druggable target that can be inhibited by certain aromatic and heterocyclic sulphonamides. Here we investigated inorganic anions and some other small molecules for their inhibition of SauBCA. The halides, nitrite, nitrate, bicarbonate, carbonate, bisulphite, sulphate, stannate, and N,N-diethyldithiocarbamate were submillimolar SauBCA inhibitors with K I s in the range of 0.26 À 0.91 mM. The most effective inhibitors were sulfamide, sulfamate, phenylboronic acid, and phenylarsonic acid with K I s of 7 À 43 mM. Several interesting inhibitors detected here may be considered lead compounds for the development of even more effective derivatives, which should be investigated for their bacteriostatic effects.

Understanding the mechanism that controls space-time coordination of elongation and division of Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB), is critical for fighting the tubercle bacillus. Most of the numerous enzymes involved in the synthesis of Mycolic acid-Arabinogalactan-Peptidoglycan complex (MAPc) in the cell wall are essential in vivo. Using a dynamic approach, we localized Mtb enzymes belonging to the fatty acid synthase-II (FAS-II) complexes and involved in mycolic acid (MA) biosynthesis in a mycobacterial model of Mtb: M. smegmatis. Results also showed that the MA transporter MmpL3 was present in the mycobacterial envelope and was specifically and dynamically accumulated at the poles and septa during bacterial growth. This localization was due to its C-terminal domain. Moreover, the FAS-II enzymes were co-localized at the poles and septum with Wag31, the protein responsible for the polar localization of mycobacterial peptidoglycan biosynthesis. The dynamic localization of FAS-II and of the MA transporter with Wag31, at the old-growing poles and at the septum suggests that the main components of the mycomembrane may potentially be synthesized at these precise foci. This finding highlights a major difference between mycobacteria and other rod-shaped bacteria studied to date. Based on the already known polar activities of envelope biosynthesis in mycobacteria, we propose the existence of complex polar machinery devoted to the biogenesis of the entire envelope. As a result, the mycobacterial pole would represent the Achilles' heel of the bacillus at all its growing stages.

Mycobacteria, including the human pathogen Mycobacterium tuberculosis, grow by inserting new cell wall material at their poles. This process and that of division are asymmetric, producing a phenotypically heterogeneous population of cells that respond non-uniformly to stress (Aldridge et al., 2012; Rego et al., 2017). Surprisingly, deletion of a single gene – lamA – leads to more symmetry, and to a population of cells that is more uniformly killed by antibiotics (Rego et al., 2017). How does LamA create asymmetry? Here, using a combination of quantitative time-lapse imaging, bacterial genetics, and lipid profiling, we find that LamA recruits essential proteins involved in cell wall synthesis to one side of the cell – the old pole. One of these proteins, MSMEG_0317, here renamed PgfA, was of unknown function. We show that PgfA is a periplasmic protein that interacts with MmpL3, an essential transporter that flips mycolic acids in the form of trehalose monomycolate (TMM), across the p...

Inorganic anions inhibit the metalloenzyme carbonic anhydrase (CA, EC 4.2.1.1) generally by coordinating to the active site metal ion. Cyanate was reported as a non-coordinating CA inhibitor but those erroneous results were subsequently corrected by another group. We review the anion CA inhibitors (CAIs) in the more general context of drug design studies and the discovery of a large number of inhibitor classes and inhibition mechanisms, including zinc binders (sulphonamides and isosteres, dithiocabamates and isosteres, thiols, selenols, benzoxaboroles, ninhydrins, etc.); inhibitors anchoring to the zinc-coordinated water molecule (phenols, polyamines, sulfocoumarins, thioxocoumarins, catechols); CAIs occluding the entrance to the active site (coumarins and derivatives, lacosamide), as well as compounds that bind outside the active site. All these new chemotypes integrated with a general procedure for obtaining isoform-selective compounds (the tail approach) has resulted, through the guidance of rigorous X-ray crystallography experiments, in the development of highly selective CAIs for all human CA isoforms with many pharmacological applications.

Emerging pharmacological strategies that target major virulence factors of antibiotic-resistant Mycobacterium tuberculosis (Mtb) are presented and discussed. This review is divided into three parts corresponding to structures and functions important for Mtb pathogenicity: the cell wall, the lipoarabinomannan, and the secretory proteins. Within the cell wall, we further focus on three biopolymeric sub-components: mycolic acids, arabinogalactan, and peptidoglycan. We present a comprehensive overview of drugs and drug candidates that target cell walls, envelopes, and secretory systems. An understanding at a molecular level of Mtb pathogenesis is provided, and potential future directions in therapeutic strategies are suggested to access new drugs to combat the growing global threat of antibiotic-resistant Mtb infection.

Mycobacterium tuberculosis enoyl-acyl-ACP reductase (InhA) has been demonstrated to be the primary target of isoniazid (INH). Recently, it was postulated that M. tuberculosis dihydrofolate reductase (DHFR) is also a target of INH, based on the findings that a 4R-INH-NADP adduct synthesized from INH by a nonenzymatic approach showed strong inhibition of DHFR in vitro , and overexpression of M. tuberculosis dfrA in M. smegmatis conferred a 2-fold increase of resistance to INH. In the present study, a plasmid expressing M. tuberculosis dfrA was transformed into M. smegmatis and M. tuberculosis strains, respectively. The transformant strains were tested for their resistance to INH. Compared to the wild-type strains, overexpression of dfrA in M. smegmatis and M. tuberculosis did not confer any resistance to INH based on the MIC values. Similar negative results were obtained with 14 other overexpressed proteins that have been proposed to bind some form of INH-NAD(P) adduct. An Escherichia...

Mycobacterium tuberculosis enoyl-acyl-ACP reductase (InhA) has been demonstrated to be the primary target of isoniazid (INH). Recently, it was postulated that M. tuberculosis dihydrofolate reductase (DHFR) is also a target of INH, based on the findings that a 4R-INH-NADP adduct synthesized from INH by a nonenzymatic approach showed strong inhibition of DHFR in vitro , and overexpression of M. tuberculosis dfrA in M. smegmatis conferred a 2-fold increase of resistance to INH. In the present study, a plasmid expressing M. tuberculosis dfrA was transformed into M. smegmatis and M. tuberculosis strains, respectively. The transformant strains were tested for their resistance to INH. Compared to the wild-type strains, overexpression of dfrA in M. smegmatis and M. tuberculosis did not confer any resistance to INH based on the MIC values. Similar negative results were obtained with 14 other overexpressed proteins that have been proposed to bind some form of INH-NAD(P) adduct. An Escherichia...

Microorganisms survive stresses by alternating the expression of genes suitable for surviving the immediate and present danger and eventually adapt to new conditions. Many bacteria have evolved a multiprotein "molecular machinery" designated the "Stressosome" that integrates different stress signals and activates alternative sigma factors for appropriate downstream responses. We and others have identified orthologs of some of the Bacillus subtilis stressosome components, RsbR, RsbS, RsbT and RsbUVW in several mycobacteria and we have previously reported mutual interactions among the stressosome components RsbR, RsbS, RsbT and RsbUVW from Mycobacterium marinum. Here we provide evidence that "STAS" domains of both RsbR and RsbS are important for establishing the interaction and thus critical for stressosome assembly. Fluorescence microscopy further suggested co-localization of RsbR and RsbS in multiprotein complexes visible as co-localized fluorescent foc...

smegmatis; wag31; stringent response; rel. The stringent response of Mycobacterium tuberculosis is coordinated by Rel and is required for full virulence in animal models. A serological-based approach identified Wag31Mtb as a protein that is upregulated in M. tuberculosis in a rel-dependent manner. This positive regulation was confirmed by analysis of M. tuberculosismRNA expression.Mycobacterium smegmatis was used to confirm that the expression of wag31Mtb from its native promoter is positively regulated by the stringent response. Furthermore, elevated wag31Mtb expression in M. smegmatis drastically alters the cell-surface hydrophobic properties.

Stimulation of AMP-activated protein kinase (AMP-PK) by ZMP (5-amino-4-imidazolecarboxamide ribotide, AICAR), formed by adenosine kinase upon addition of AICAriboside to isolated rat hepatocytes, results in inhibition of fatty acid and cholesterol synthesis by inactivation of acetyl-CoA carboxylase and 3-hydroxy-3-methylglutaryl-CoA reductase, respectively (Henin et al. (1995) FASEB J. 9, 541-546). The effects of ZMP and other AMP analogues have now been compared with those of AMP on AMP-PK purified from rat liver. ZMP stimulated AMP-PK to the same maximal extent as AMP (about 10-fold). ZMP had less affinity for AMP-PK than AMP, but this affinity was similarly influenced by ATP: half-maximal effects, requiring 0.4 mM AMP or 5 mM ZMP at 3 mM ATP, were obtained with 9 txM AMP or 0.4 mM ZMP at 0.2 mM ATP. The kinetic parameters of AMP-PK for the SAMS peptide and for ATP were influenced in the same way by ZMP and AMP. Stimulation of AMP-PK by ZMP was additive with AMP, up to when maximal stimulation was obtained. Taken together, these results indicate that ZMP binds to the same site as AMP on AMP-PK. Tubercidin 5'-monophosphate, 2'-deoxy-AMP and Ara-AMP stimulated AMP-PK, but N6-methyl-AMP, 1,N6-etheno-AMP, 6-mercaptopurine riboside 5'-monophosphate, adenylosuccinate and succinyl-AICAR were ineffective, suggesting that a free 6-NH 2 group may be important for binding of effectors to AMP-PK.

We have sequenced two tryptic/chymotryptic peptides (TC3 and TC3a) containing a third site phosphorylated on rat acetyl-CoA carboxylase by the AMP-activated protein kinase. Comparison with the complete sequence of rat acetyl-CoA carboxylase predicted from the cDNA sequence [L6pez-Casillas et al. (I 988) Proc. Nut1 Acad. Sci. USA 85, 5784-57881 shows that this site corresponds to Ser1215. 2. Comparison of the cDNA sequence with previous amino acid sequence data identifies the other two sites for the AMP-activated protein kinase as Ser79 and Ser1200. A total of eight serine residues phosphorylated in vitro by six protein kinases can now be identified: six of these (Ser23, Ser25, Ser29, Ser77, Ser79 and Ser95) are clustered in the amino terminal region, while two (Ser1200 and Ser1215) are located in the central region. 3. Prior phosphorylation of Ser77 and Ser1200 by cyclic-AMP-dependent protein kinase prevents subsequent phosphorylation of Ser79 and Ser1200, but not Ser1215, by the AMP-activated protein kinase. Phosphorylation of Ser1215 under these conditions is not associated with a change in enzyme activity. 4. Limited trypsin treatment of native acetyl-CoA carboxylase selectively cleaves off the highly phosphorylated amino-terminal region containing Ser79. 5. Phosphorylation at Ser79 and Ser1200 by the AMP-activated protein kinase dramatically decreases V,,, and increases the A0.5 for citrate. Phosphorylation at Ser77 and Ser1200 by cyclic-AMP-dependent protein kinase causes more modest changes in the Ao.s for citrate and the V,,,. Dephosphorylation, or removal of the aminoterminal region containing Ser77/79 using trypsin, reverses all of these effects. 6. These results suggest that the effects of the AMP-activated protein kinase on acetyl-CoA carboxylase activity are mediated entirely by phosphorylation of Ser79, and not Ser1200 and Serl215. The smaller effects of cyclic-AMP-dependent protein kinase are mediated by phosphorylation of Ser77.

EJB 89 0546 1. We have synthesized two peptides, one based on the exact sequence around the unique site (Ser79) for thc AMP-activated protein kinase on rat acetyl-CoA carboxylase (SSMS peptide) and another in which the serine residue corresponding to the site for cyclic-AMP-dependent protein kinase (Ser77) was replaced by alanine (SAMS peptide). 2. Both peptides were phosphorylated with similar kinetics by the AMP-activated protein kinase, but only the SSMS peptide was a substrate for cyclic-AMP-dependent protein kinase. The SAMS peptide was not phosphorylated by any of five other purified protein kinases tested. 3. The K,,, of AMP-activated protein kinase for the SAMS peptidc is higher than that for acetyl-CoA carboxylase, but the V,,, for peptide phosphorylation is 2.5 times higher than that of its parent protein. This peptide therefore gives a convenient and sensitive assay for the AMP-activated protein kinase. 4. Acetyl-CoA-carboxylase kinase and peptide kinase activities copurify through six steps from a post-mitochondria1 supernatant of rat liver, showing that the SAMS peptide is a specific substrate for the AMPaclivated protein kinasc in this tissuc. Wc could not demonstrate AMP-depcndence of the kinase activity in crude preparations, apparently due to endogenous AMP remaining bound to the enzyme. However, 8-bromoadenosine 5-monophosphate (Br8AMP) is a partial agonist at the allosteric (AMP) site, and inhibition by 2 mM Br'AMP can be used to test that one is measuring the AMP-stimulated form of the kinase. 5. Using this approach, we have examined the kinase activity in nine different rat tissues, plus a mousc macrophage cell line, iind find that there is a correlation betwcen tissucs expressing significant levels of peptide kinase activity and those active in the synthesis or storage of lipids. 6. We also use the peptide assay to show that cyclic AMP-dependent protein kinase does not activate purified AMP-activated protein kinase, and does not affect the activation of partially purified AMP-activated protein kinase by endogenous kinase kinase. The AMP-activated protein kinase is a recently recognized multisubstrate kinase which appears to play a central role in regulation of fatty acid and cholesterol metabolism [l]. The kinase was originally recognized for its ability to inactivate 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase [2], but has recently also been shown to phosphorylate and inactivate acetyl-CoA carboxylase [3,4]. and to phosphorylatc hormone-sensitive lipase which blocks activation of that enzyme by cyclic-AMP-dependent protein kinase [5]. The kinase is activated by AMP, and also by phosphorylation catalyzed by a distinct 'kinase kinase' [3]. The existing assays for this kinase either involve its ability to inactivate microsomal HMG-CoA reductase in a time-and

Stimulation of AMP-activated protein kinase (AMP-PK) by ZMP (5-amino-4-imidazolecarboxamide ribotide, AICAR), formed by adenosine kinase upon addition of AICAriboside to isolated rat hepatocytes, results in inhibition of fatty acid and cholesterol synthesis by inactivation of acetyl-CoA carboxylase and 3-hydroxy-3-methylglutaryl-CoA reductase, respectively (Henin et al. (1995) FASEB J. 9, 541–546). The effects of ZMP and other AMP analogues have now been

AMP-activated protein kinase is a multisubstrate protein kinase that, in liver, inactivates both acetyl-CoA carboxylase, the rate-limiting enzyme of fatty acid synthesis, and 3-hydroxy-3-methyl-glutaryl-CoA reductase, the rate-limiting enzyme of cholesterol synthesis. AICAR (5-amino 4-imidazolecarboxamide ribotide, ZMP) was found to stimulate up to 10-fold rat liver AMP-activated protein kinase, with a half-maximal effect at approximately 5 mM. In accordance with previous observations, addition to suspensions of isolated rat hepatocytes of 50-500 microM AICAriboside, the nucleoside corresponding to ZMP, resulted in the accumulation of millimolar concentrations of the latter. This was accompanied by a dose-dependent inactivation of both acetyl-CoA carboxylase and 3-hydroxy-3-methylglutaryl-CoA reductase. Addition of 50-500 microM AICAriboside to hepatocyte suspensions incubated in the presence of various substrates, including glucose and lactate/pyruvate, caused a parallel inhibition...

The elevation of adenosine levels induced by anoxia in isolated rat hepatocytes has been shown to result mainly from an arrest of the recycling of the nucleoside by adenosine kinase [Bontemps, Vincent and Van den Berghe (1993) Biochem. J. 290, 671-677]. To assess the activity of the latter enzyme in intact hepatocytes, incorporation of radioactive adenosine into the cells' adenine nucleotides was measured. Unexpectedly, despite the near-absence of ATP in anoxic cells, 40% of 50 microM [8-14C]adenosine was still incorporated into adenylates over 5 min. Moreover, whereas unlabelled and labelled adenosine were utilized in parallel in normoxic cells, uptake of [8-14C]adenosine did not correspond to a net disappearance of adenosine in anoxic cells. Addition of 1 mM unlabelled adenosine to anoxic hepatocytes in which the adenine nucleotides had been prelabelled with [U-14C]adenine induced an immediate loss of their radioactivity. The latter was recovered in the form of adenosine, but ...

5-Amino-4-imidazolecarboxamide riboside (AICAriboside; Z-riboside), the nucleotide corresponding to AICAribotide (AICAR or ZMP), an intermediate of the 'de novo' pathway of purine nucleotide biosynthesis, has been shown to inhibit gluconeogenesis in isolated rat hepatocytes [Vincent, Marangos, Gruber & Van den Berghe (1991) Diabetes 40, 1259-1266]. We now report that glycosis is also inhibited and even more sensitive to AICAriboside in these cells. In hepatocyte suspensions from fasted rats, production of lactate from 15 mM-glucose was half-maximally inhibited by 25-50 microM-AICAriboside. AICAriboside influenced two regulatory steps of glycolysis: (1) it decreased the release of 3H2O from [2-3H]glucose and the concentrations of both glucose 6-phosphate and fructose 6-phosphate, indicating that it diminished the phosphorylation of glucose by glucokinase; (2) it decreased the concentration of fructose 2,6-bisphosphate (Fru-2,6-P2), the main physiological stimulator of liver 6...

Stimulation of AMP-activated protein kinase (AMP-PK) by ZMP (5-amino-4-imidazolecarboxamide ribotide, AICAR), formed by adenosine kinase upon addition of AICAriboside to isolated rat hepatocytes, results in inhibition of fatty acid and cholesterol synthesis by inactivation of acetyl-CoA carboxylase and 3-hydroxy-3-methylglutaryl-CoA reductase, respectively (Henin et al. (1995) FASEB J. 9, 541–546). The effects of ZMP and other AMP analogues have now been

Mycolic acids (MAs) have a strategic location within the mycobacterial envelope, deeply influencing its architecture and permeability, and play a determinant role in the pathogenicity of mycobacteria. The fatty acid synthase type II (FAS-II) multienzyme system is involved in their biosynthesis. A combination of pull-downs and proteomics analyses led to the discovery of a mycobacterial protein, HadD, displaying highly specific interactions with the dehydratase HadAB of FAS-II. In vitro activity assays and homology modeling showed that HadD is, like HadAB, a hot dog folded (R)-specific hydratase/ dehydratase. A hadD knockout mutant of Mycobacterium smegmatis produced only the medium-size alpha'-MAs. Data strongly suggest that HadD is involved in building the third meromycolic segment during the late FAS-II elongation cycles, leading to the synthesis of the full-size alpha-and epoxy-MAs. The change in the envelope composition induced by hadD inactivation strongly altered the bacterial fitness and capacities to aggregate, assemble into colonies or biofilms and spread by sliding motility, and conferred a hypersensitivity to the firstline antimycobacterial drug rifampicin. This showed that the cell surface properties and the envelope integrity were greatly affected. With the alarmingly increasing case number of nontuberculous mycobacterial diseases, HadD appears as an attractive target for drug development. Mycobacteria are responsible for a large diversity of infectious diseases. The best known is tuberculosis (TB) since it represents one of the leading causes of death worldwide 1. The emergence of multidrug and extensively drug-resistant Mycobacterium tuberculosis strains has challenged TB control 1. The nontuberculous mycobacteria (NTM), such as M. smegmatis, M. kansasii, M. abscessus, and members of the M. avium and M. fortuitum/ chelonae complexes, form a heterogeneous group of environmental organisms found in water, soil, dust, biofilms and diverse animals 2. Pathogenic NTM can cause infectious diseases in livestock and wildlife, as well as human diseases, such as pulmonary and disseminated diseases, lymphadenitis, and skin and soft tissue lesions 2,3. Like M. tuberculosis, they constitute a major cause of opportunistic infections in HIV patients 4. Alarmingly, the number of reported cases has been increasing worldwide 2,5. NTM also colonize man-made environments and cause healthcare-associated infections, which are a major public health concern 2-4. NTM are intrinsically resistant to most conventionally utilized antimicrobials and many of the antituberculous drugs under development are inactive against NTM 4,5. Thus, novel molecular strategies for eradicating NTM from infection sources and hosts are urgently needed 5 .

AMP-activated protein kinase, purified from rat liver as far as the diethylaminoethyl-Sepharose step, is inactivated by treatment with protein phosphatase 2C, and reactivated by an endogenous 'kinase kinase'. Further purification of AMP-activated protein kinase on Blue Sepharose removes the kinase kinase, but the system can be reconstituted by adding back the flow-through from the Blue-Sepharose column. The kinase kinase can be further purified by subjecting the flow-through from the Blue-Sepharose column to chromatography on a Mono-Q column. A single peak of kinase kinase activity is obtained. Using this fraction, and the most highly purified preparation of AMPactivated protein kinase, phosphorylation of the 63-kDa polypeptide, previously identified as the catalytic subunit of AMP-activated protein kinase, can be demonstrated. As previously shown in the partially purified system, phosphorylation of the 63-kDa polypeptide is markedly stimulated by AMP. The kinase and kinase kinase reactions exhibit similar dependence on AMP concentration. The structurally related AMP analogue, 8-aza-9-deazaadenosine-5'-monophosphate, mimics the effect of AMP on both allosteric activation and phosphorylation of the kinase, while adenosine (5')tetraphospho(5')adenosine antagonizes both effects. These results suggest that both the allosteric effect of AMP, and the promotion of phosphorylation and activation by the kinase kinase, are due to binding of AMP to a single site on the kinase. Mammalian AMP-activated protein kinase (AMP-PK) is now recognized as a multisubstrate protein kinase which phosphorylates and inactivates at least three key regulatory enzymes of lipid metabolism, i.e. 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase (cholesterol synthesis), acetyl-CoA carboxylase (fatty acid synthesis) and hormonesensitive lipase (triacylglyceroVcholestero1-ester breakdown) [I, 21. This may be an ancient and highly conserved regulatory system, because there is a protein kinase in higher plants which appears to be a homologue of AMP-PK by several functional criteria, including inactivation of HMG-CoA reductase, although it is not activated by AMP [3]. Mammalian AMP-PK is allosterically activated up to fivefold by physiological concentrations of AMP [4], and we have presented

Understanding the mechanism that controls space-time coordination of elongation and division of Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB), is critical for fighting the tubercle bacillus. Most of the numerous enzymes involved in the synthesis of Mycolic acid-Arabinogalactan-Peptidoglycan complex (MAPc) in the cell wall are essential in vivo. Using a dynamic approach, we localized Mtb enzymes belonging to the fatty acid synthase-II (FAS-II) complexes and involved in mycolic acid (MA) biosynthesis in a mycobacterial model of Mtb: M. smegmatis. Results also showed that the MA transporter MmpL3 was present in the mycobacterial envelope and was specifically and dynamically accumulated at the poles and septa during bacterial growth. This localization was due to its C-terminal domain. Moreover, the FAS-II enzymes were co-localized at the poles and septum with Wag31, the protein responsible for the polar localization of mycobacterial peptidoglycan biosynthesis. The dynamic localization of FAS-II and of the MA transporter with Wag31, at the old-growing poles and at the septum suggests that the main components of the mycomembrane may potentially be synthesized at these precise foci. This finding highlights a major difference between mycobacteria and other rod-shaped bacteria studied to date. Based on the already known polar activities of envelope biosynthesis in mycobacteria, we propose the existence of complex polar machinery devoted to the biogenesis of the entire envelope. As a result, the mycobacterial pole would represent the Achilles' heel of the bacillus at all its growing stages.

The first-line specific antituberculous drug isoniazid inhibits the fatty acid elongation system (FAS) FAS-II involved in the biosynthesis of mycolic acids, which are major lipids of the mycobacterial envelope. The MabA protein that catalyzes the second step of the FAS-II elongation cycle is structurally and functionally related to the in vivo target of isoniazid, InhA, an NADH-dependent enoyl-acyl carrier protein reductase. The present work shows that the NADPH-dependent β-ketoacyl reduction activity of MabA is efficiently inhibited by isoniazid in vitro by a mechanism similar to that by which isoniazid inhibits InhA activity. It involves the formation of a covalent adduct between MnIII-activated isoniazid and the MabA cofactor. Liquid chromatography-mass spectrometry analyses revealed that the isonicotinoyl-NADP adduct has multiple chemical forms in dynamic equilibrium. Both kinetic experiments with isolated forms and purification of the enzyme-ligand complex strongly suggested th...

Mycobacteria are well known for their taxonomic diversity, their impact on global health, and for their atypical cell wall and envelope. In addition to a cytoplasmic membrane and a peptidoglycan layer, the cell envelope of members of the order Corynebacteriales, which include Mycobacterium tuberculosis, also have an arabinogalactan layer connecting the peptidoglycan to an outer membrane, the so-called "mycomembrane." This unusual cell envelope composition of mycobacteria is of prime importance for several physiological processes such as protection from external stresses and for virulence. Although there have been recent breakthroughs in the elucidation of the composition and organization of this cell envelope, its evolutionary origin remains a mystery. In this perspectives article, the characteristics of the cell envelope of mycobacteria with respect to other actinobacteria will be dissected through a molecular evolution framework in order to provide a panoramic view of the evolutionary pathways that appear to be at the origin of this unique cell envelope. In combination with a robust molecular phylogeny, we have assembled a gene matrix based on the presence or absence of key determinants of cell envelope biogenesis in the Actinobacteria phylum. We present several evolutionary scenarios regarding the origin of the mycomembrane. In light of the data presented here, we also propose a novel alternative hypothesis whereby the stepwise acquisition of core enzymatic functions may have allowed the sequential remodeling of the external cell membrane during the evolution of Actinobacteria and has led to the unique mycomembrane of slow-growing mycobacteria as we know it today.

Aim: There is a necessity for new drugs to be more efficient than today's standard due to the emergence of drug-resistant strains of Mycobacterium tuberculosis (Mtb) Results/methodology: 12 new isoniazid-based adamantane derivatives were synthesized and tested for their antitubercular activity. The pharmacological test results and the aqueous dissolution profile of representative examples of the new molecules are in agreement with the computational results obtained from docking poses and molecular dynamics simulations on the tested compounds. Conclusion: Among their congeners, the adamantane isonicotinoyl hydrazones Ia and Ih exhibit the best antitubercular activity (MIC = 0.04 μg/ml) and the lowest cytotoxicity (selectivity index ≥2500). These results are useful for in future in vivo studies. Graphical abstract: Z XCH=NNHC N H XCH=NNHC N O Y CONHN=CH Q V O CHCONHN=CH N Ia-e,h If,g IIa-c IId X, Z : H; (CH 2) n , n = 0, 1; phenyl; cyclopentyl Y, V, Q : H, N, CONHN=CHPy Ia : MIC = 0.04 μg/ml

A highly purified rat liver protein kinase phosphorylates and inactivates acetyl-CoA carboxylase, and causes rapid inactivation of microsomal HMG-CoA reductase in the presence of MgATP. Both effects are stimulated in an identical manner by AMP, and are greatly reduced by prior treatment of the kinase with purified protein phosphatase. The dephosphorylated kinase can be reactivated in the presence of MgATP, apparently due to a distinct kinase kinase, and this reactivation is stimulated by nanomolar concentrations of palmitoyl-CoA. These results show that a common, bicyclic protein kinase cascade can potently inactivate the regulatory enzymes of both fatty acid and cholesterol biosynthesis.

1. We have purified the AMP-activated protein kinase 4800-fold from rat liver. The acetyl-CoA carboxylase kinase and 3-hydroxy-3-methylglutaryl-CoA(HMG-CoA) reductase kinase activities copurify through all six purification steps and are inactivated with similar kinetics by treatment with the reactive ATP analogue fluorosulphonylbenzoyladenosine.

A highly purified rat liver protein kinase phosphorylates and inactivates acetyl-CoA carboxylase, and causes rapid inactivation of microsomal HMG-CoA reductase in the presence of MgATP. Both effects are stimulated in an identical manner by AMP, and are greatly reduced by prior treatment of the kinase with purified protein phosphatase. The dephosphorylated kinase can be reactivated in the presence of MgATP, apparently due to a distinct kinase kinase, and this reactivation is stimulated by nanomolar concentrations of palmitoyl-CoA. These results show that a common, bicyclic protein kinase cascade can potently inactivate the regulatory enzymes of both fatty acid and cholesterol biosynthesis.

Oral supplementation of 10 mmol/kg/day of D-ribose to a patient with an inherited deficit of adenylosuccinase, severe psychomotor retardation, and epilepsy caused a marked increase in plasma concentration and urinary excretion of urate, while minor changes in succinylpurine levels were observed. D-Ribose administration was accompanied by a slight improvement of behaviour and a progressive reduction of seizure frequency, which increased dramatically upon two attempts to withdraw the drug. Substitution of D-ribose with an equivalent amount of D-glucose did not result in an increase of seizure frequency. ß

Oral supplementation of 10 mmol/kg/day of D-ribose to a patient with an inherited deficit of adenylosuccinase, severe psychomotor retardation, and epilepsy caused a marked increase in plasma concentration and urinary excretion of urate, while minor changes in succinylpurine levels were observed. D-Ribose administration was accompanied by a slight improvement of behaviour and a progressive reduction of seizure frequency, which increased dramatically upon two attempts to withdraw the drug. Substitution of D-ribose with an equivalent amount of D-glucose did not result in an increase of seizure frequency. ß

The role of carbonic anhydrase in de novo lipid synthesis was examined by measuring [1-14C]acetate incorporation into total lipids, fatty acids and non-saponifiable lipids in freshly isolated rat hepatocytes. Two carbonic anhydrase inhibitors, trifluoromethylsulphonamide (TFMS) and ethoxozolamide (ETZ) decreased incorporation of 14C into total lipids. Both fatty acid and non-saponifiable lipid components of the total lipid were inhibited to approximately the same extent by 100 microM TFMS (29 +/- 0.3% and 35 +/- 0.3% of control respectively in replicate studies). However, neither drug significantly affected ATP concentrations or the transport activity of Na+/K(+)-ATPase, two measures of cell viability. To establish the site of this inhibition, water-soluble 14C-labelled metabolites from perchloric acid extracts of the radiolabelled cells were separated by ion-exchange chromatography. TFMS inhibited 14C incorporation into citrate, malate, alpha-oxoglutarate and fumarate, but had no e...

Understanding the mechanism that controls space-time coordination of elongation and division of Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB), is critical for fighting the tubercle bacillus. Most of the numerous enzymes involved in the synthesis of Mycolic acid -Arabinogalactan-Peptidoglycan complex (MAPc) in the cell wall are essential in vivo. Using a dynamic approach, we localized Mtb enzymes belonging to the fatty acid synthase-II (FAS-II) complexes and involved in mycolic acid (MA) biosynthesis in a mycobacterial model of Mtb: M. smegmatis. Results also showed that the MA transporter MmpL3 was present in the mycobacterial envelope and was specifically and dynamically accumulated at the poles and septa during bacterial growth. This localization was due to its C-terminal domain. Moreover, the FAS-II enzymes were co-localized at the poles and septum with Wag31, the protein responsible for the polar localization of mycobacterial peptidoglycan biosynthesis. The dynamic localization of FAS-II and of the MA transporter with Wag31, at the old-growing poles and at the septum suggests that the main components of the mycomembrane may potentially be synthesized at these precise foci. This finding highlights a major difference between mycobacteria and other rod-shaped bacteria studied to date. Based on the already known polar activities of envelope biosynthesis in mycobacteria, we propose the existence of complex polar machinery devoted to the biogenesis of the entire envelope. As a result, the mycobacterial pole would represent the Achilles' heel of the bacillus at all its growing stages. Citation: Carel C, Nukdee K, Cantaloube S, Bonne M, Diagne CT, et al. (2014) Mycobacterium tuberculosis Proteins Involved in Mycolic Acid Synthesis and Transport Localize Dynamically to the Old Growing Pole and Septum. PLoS ONE 9(5): e97148.

1. We have purified the AMP-activated protein kinase 4800-fold from rat liver. The acetyl-CoA carboxylase kinase and 3-hydroxy-3-methylglutaryl-CoA(HMG-CoA) reductase kinase activities copurify through all six purification steps and are inactivated with similar kinetics by treatment with the reactive ATP analogue fluorosulphonylbenzoyladenosine.

The (R)-specific 3-hydroxyacyl dehydratases/trans-enoyl hydratases are key proteins in the biosynthesis of fatty acids. In mycobacteria, such
enzymes remain unknown, although they are involved in the biosynthesis of major and essential lipids like mycolic acids. First bioinformatic
analyses allowed to identify a single candidate protein, namely Rv3389c, that belongs to the hydratases 2 family and is most likely made of a
distinctive asymmetric double hot dog fold. The purified recombinant Rv3389c protein was shown to efficiently catalyze the hydration of (C8–
C16) enoyl-CoA substrates. Furthermore, it catalyzed the dehydration of a 3-hydroxyacyl-CoA in coupled reactions with both reductases (MabA
and InhA) of the acyl carrier protein (ACP)-dependent M. tuberculosis fatty acid synthase type II involved in mycolic acid biosynthesis. Yet, the
facts that Rv3389c activity decreased in the presence of ACP, versus CoA, derivative and that Rv3389c knockout mutant had no visible variation
of its fatty acid content suggested the occurrence of additional hydratase/dehydratase candidates. Accordingly, further and detailed bioinformatic
analyses led to the identification of other members of the hydratases 2 family in M. tuberculosis.

The first-line specific antituberculous drug isoniazid inhibits the fatty acid elongation system (FAS) FAS-II
involved in the biosynthesis of mycolic acids, which are major lipids of the mycobacterial envelope. The MabA
protein that catalyzes the second step of the FAS-II elongation cycle is structurally and functionally related to
the in vivo target of isoniazid, InhA, an NADH-dependent enoyl-acyl carrier protein reductase. The present
work shows that the NADPH-dependent
-ketoacyl reduction activity of MabA is efficiently inhibited by
isoniazid in vitro by a mechanism similar to that by which isoniazid inhibits InhA activity. It involves the
formation of a covalent adduct between MnIII-activated isoniazid and the MabA cofactor. Liquid chromatography-
mass spectrometry analyses revealed that the isonicotinoyl-NADP adduct has multiple chemical forms
in dynamic equilibrium. Both kinetic experiments with isolated forms and purification of the enzyme-ligand
complex strongly suggested that the molecules active against MabA activity are the oxidized derivative and a
major cyclic form. Spectrofluorimetry showed that the adduct binds to the MabA active site. Modeling of the
MabA-adduct complex predicted an interaction between the isonicotinoyl moiety of the inhibitor and Tyr185.
This hypothesis was supported by the fact that a higher 50% inhibitory concentration of the adduct was
measured for MabA Y185L than for the wild-type enzyme, while both proteins presented similar affinities for
NADP. The crystal structure of MabA Y185L that was solved showed that the substitution of Tyr185 induced
no significant conformational change. The description of the first inhibitor of the
-ketoacyl reduction step of
fatty acid biosynthesis should help in the design of new antituberculous drugs efficient against multidrugresistant
tubercle bacilli.

Despite the existence of efficient chemotherapy,
tuberculosis remains a leading cause of mortality
worldwide. New drugs are urgently needed to reduce
the potential impact of the emergence of multidrugresistant
strains of the causative agent
Mycobacterium
tuberculosis
(
Mtb
). The front-line antibiotic
isoniazid (INH), and several other drugs, target the
biosynthesis of mycolic acids and especially the
Fatty Acid Synthase-II (FAS-II) elongation system.
This biosynthetic pathway is essential and specific
for mycobacteria and still represents a valuable system
for the search of new anti-tuberculous agents.
Several data, in the literature, suggest the existence
of protein–protein interactions within the FAS-II system.
These interactions themselves might serve as
targets for a new generation of drugs directed against
Mtb
. By using an extensive
in vivo
yeast two-hybrid
approach and
in vitro
co-immunoprecipitation, we
have demonstrated the existence of both homotypic
and heterotypic interactions between the known components
of FAS-II. The condensing enzymes KasA,
KasB and mtFabH interact with each other and with
the reductases MabA and InhA. Furthermore, we have
designed and constructed point mutations of the
FAS-II reductase MabA, able to disrupt its homotypic
interactions and perturb the interaction pattern of
this protein within FAS-II. Finally, we showed by a
transdominant genetic approach that these mutants
are dominant negative in both non-pathogenic and
pathogenic mycobacteria. These data allowed us to
draw a dynamic model of the organization of FAS-II.

Understanding the mechanism that controls space-time coordination of elongation and division of Mycobacterium
tuberculosis (Mtb), the causative agent of tuberculosis (TB), is critical for fighting the tubercle bacillus. Most of the numerous
enzymes involved in the synthesis of Mycolic acid - Arabinogalactan-Peptidoglycan complex (MAPc) in the cell wall are
essential in vivo. Using a dynamic approach, we localized Mtb enzymes belonging to the fatty acid synthase-II (FAS-II)
complexes and involved in mycolic acid (MA) biosynthesis in a mycobacterial model of Mtb: M. smegmatis. Results also
showed that the MA transporter MmpL3 was present in the mycobacterial envelope and was specifically and dynamically
accumulated at the poles and septa during bacterial growth. This localization was due to its C-terminal domain. Moreover,
the FAS-II enzymes were co-localized at the poles and septum with Wag31, the protein responsible for the polar localization
of mycobacterial peptidoglycan biosynthesis. The dynamic localization of FAS-II and of the MA transporter with Wag31, at
the old-growing poles and at the septum suggests that the main components of the mycomembrane may potentially be
synthesized at these precise foci. This finding highlights a major difference between mycobacteria and other rod-shaped
bacteria studied to date. Based on the already known polar activities of envelope biosynthesis in mycobacteria, we propose
the existence of complex polar machinery devoted to the biogenesis of the entire envelope. As a result, the mycobacterial
pole would represent the Achilles’ heel of the bacillus at all its growing stages.

Cyclic AMP (300 ~M) activates phosphofructokinase from dialyzed haemolysates of mature rat erythrocytes. The main conclusions are: a) Cyclic AMP, at pH 7.1 and low concentrations of fructose-6-phosphate, is able to reverse the inhibition produced by different amounts of ATP (up to 1.5 mM). b) The cyclic nucleotide is a positive allosteric effector of the enzyme as shown by the displacement of sigmoidal fructose-6-phosphate saturation curve to hyperbolic kinetics in the presence of inhibitory concentrations (1.5 mM) of ATP. c) Cyclic AMP has no significant influence as deinhibitor of phosphofructokinase either at pH 7.1 and noninhibitory levels (0.25 mM) of ATP or at pH 8.1 and inhibitory (1.5 mM) or non-inhibitory (0.25 mM) concentrations of ATP. Similar conclusions were obtained with 300/XM AMP but not at a lower concentration (3/XM) with both nucleotides.

Rv0241c (HtdX) is a putative (3R)-hydroxyacyl-CoA dehydratase of Mycobacterium tuberculosis. The htdX gene belongs to a conserved operon and is expressed in mycobacteria in the presence of several fatty-acid synthase II drugs. To elucidate the structure of HtdX, the protein was cloned, overexpressed, purified to homogeneity and crystallized. The protein was crystallized from two conditions: (i) 3 M sodium chloride, 0.1 M Na HEPES pH 8.0 and (ii) 2.5 M sodium chloride, 0.1 M Tris-HCl pH 8.5. A complete diffraction data set was collected from crystals from both conditions. The crystal from the first condition diffracted to 2.3 Å resolution and belonged to space group I4 1 , with unit-cell parameters a = b = 61.51, c = 143.81 Å . Crystals from the second condition diffracted to 3.

Stimulation of AMP-activated protein kinase (AMP-PK) by ZMP (5-amino-4-imidazolecarboxamide ribotide, AICAR), formed by adenosine kinase upon addition of AICAriboside to isolated rat hepatocytes, results in inhibition of fatty acid and cholesterol synthesis by inactivation of acetyl-CoA carboxylase and 3-hydroxy-3-methylglutaryl-CoA reductase, respectively (Henin et al. (1995) FASEB J. 9, 541-546). The effects of ZMP and other AMP analogues have now been compared with those of AMP on AMP-PK purified from rat liver. ZMP stimulated AMP-PK to the same maximal extent as AMP (about 10-fold). ZMP had less affinity for AMP-PK than AMP, but this affinity was similarly influenced by ATP: half-maximal effects, requiring 0.4 mM AMP or 5 mM ZMP at 3 mM ATP, were obtained with 9 txM AMP or 0.4 mM ZMP at 0.2 mM ATP. The kinetic parameters of AMP-PK for the SAMS peptide and for ATP were influenced in the same way by ZMP and AMP. Stimulation of AMP-PK by ZMP was additive with AMP, up to when maximal stimulation was obtained. Taken together, these results indicate that ZMP binds to the same site as AMP on AMP-PK. Tubercidin 5'-monophosphate, 2'-deoxy-AMP and Ara-AMP stimulated AMP-PK, but N6-methyl-AMP, 1,N6-etheno-AMP, 6-mercaptopurine riboside 5'-monophosphate, adenylosuccinate and succinyl-AICAR were ineffective, suggesting that a free 6-NH 2 group may be important for binding of effectors to AMP-PK.