The product of the gene ponA present in cosmid MTCY21D4, one of the collection of clones represen... more The product of the gene ponA present in cosmid MTCY21D4, one of the collection of clones representing the genome of Mycobacterium tuberculosis, has been named penicillin-binding protein 1* (PBP1*), by analogy to the previously characterized PBP1* of M. leprae. This gene has been overexpressed in Escherichia coli. His '-tagged PBP1* localizes to the membranes of induced E. coli cells. Its susceptibility to degradation upon proteinase K digestion of spheroplasts from E. coli expressing the protein supports the view that the majority of the protein translocates to the periplasmic side of the membrane. Recombinant PBP1* binds benzylpenicillin and several other β-lactams, notably cefotaxime, with high affinity. Truncation of the Nterminal 64 amino acid residues results in an expressed protein
Current trends on biotechnology & microbiology, Sep 6, 2019
Antimicrobial resistance (AMR) is a growing problem worldwide. Resistance to antibiotics can occu... more Antimicrobial resistance (AMR) is a growing problem worldwide. Resistance to antibiotics can occur in a number of ways, one of which is removal of the drugs from the cell via efflux pump macromolecular machineries. As such, efflux pumps can provide a background level of resistance to many different classes of antimicrobials and are a major contributor to AMR. Inhibition of efflux pumps therefore has the potential to reverse resistance to many antibiotics in one go and is an attractive potential for treating resistant infections. Whilst a number of efflux inhibitors are known, none are currently used clinically due to harmful side effects. Development of novel inhibitors is therefore imperative. The article aims to review accumulation assays and efflux assays, two of the most common laboratory techniques used to identify and characterise candidate efflux inhibitors.
Strains of members of Enterobacteriaceae, namely Escherichia coli (18), Klebsiella aerogenes (16)... more Strains of members of Enterobacteriaceae, namely Escherichia coli (18), Klebsiella aerogenes (16), and Serratia marcescens (16) were screened for Cd resistance or sensitivity. Only one strain each of these was resistant to high levels (25 n moles/0.05 ml) CdCl2. The Minimal inhibitory concentration (MIC) of sensitive strains ranged from 0.8-5 micrograms/ml. All the resistant strains were simultaneously resistant to a number of antibiotics. Treatment with sodium dodecyl sulfate eliminated resistance to Cd and to some antibiotics.
This article is an open access article distributed under the terms and conditions of the Creative... more This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY
This is a PDF file of an article that has undergone enhancements after acceptance, such as the ad... more This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
From Allium stipitatum, three pyridine-N-oxide alkaloids (1-3) possessing disulfide functional gr... more From Allium stipitatum, three pyridine-N-oxide alkaloids (1-3) possessing disulfide functional groups were isolated. The structures of these natural products were elucidated by spectroscopic means as 2-(methyldithio)pyridine-N-oxide (1), 2-[(methylthiomethyl)dithio]pyridine-N-oxide (2), and 2,2′-dithio-bis-pyridine-N-oxide (3). The proposed structure of 1 was confirmed by synthetic Smethylthiolation of commercial 2-thiopyridine-N-oxide. Compounds 1 and 2 are new natural products, and 3 is reported for the first time from an Allium species. All compounds were evaluated for activity against fast-growing species of Mycobacterium, methicillin-resistant Staphylococcus aureus, and a multidrug-resistant (MDR) variants of S. aureus. Compounds 1 and 2 exhibited minimum inhibitory concentrations (MICs) of 0.5-8 μg/mL against these strains. A small series of analogues of 1 were synthesized in an attempt to optimize antibacterial activity, although the natural product had the most potent in vitro activity. In a whole-cell assay at 30 μg/mL, 1 was shown to give complete inhibition of the incorporation of 14 C-labeled acetate into soluble fatty acids, indicating that it is potentially an inhibitor of fatty acid biosynthesis. In a human cancer cell line antiproliferative assay, 1 and 2 displayed IC 50 values ranging from 0.3 to 1.8 μM with a selectivity index of 2.3 when compared to a human somatic cell line. Compound 1 was evaluated in a microarray analysis that indicated a similar mode of action to menadione and 8-quinolinol by interfering with the thioredoxin system and up-regulating the production of various heat shock proteins. This compound was also assessed in a mouse model for in vivo toxicity. ¶ Dedicated to Dr. David G. I. Kingston of Virginia Polytechnic Institute and State University for his pioneering work on bioactive natural products.
This article is an open access article distributed under the terms and conditions of the Creative... more This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY
Journal of Infectious Diseases and Therapy, Sep 2, 2015
T uberculosis (TB), having been declared a global emergency since 1993, is still an issue of grea... more T uberculosis (TB), having been declared a global emergency since 1993, is still an issue of great public health concern because of the emergence of multi-and extensively-drug-resistant strains of Mycobacterium tuberculosis. New molecules with pleiotropic modes of action are urgently required to tackle this challenging menace. Pyridine-N-oxide alkaloids possessing disulfide functional groups were isolated from the bulbs of Allium stipitatum, belonging to the genus Allium with common members like garlic, onion, leeks and chives. From this discovery, a series of methyldisulfides were synthesized based on the structure of the natural product disulphides isolated from the bulbs of Allium stipitatum. The synthetic analogues were produced by adopting the method of Kitson and Loomes, briefly, the appropriate aromatic thiol purchased from Sigma Aldrich was S-methylthiolated using S-methyl methanethiosulphonate and back extracted with dichloromethane. Antibacterial activities using various high-throughput whole cell phenotypic assays were carried out. A selection of five compounds of that chemical class showed antimycobacterial activities at clinically relevant concentrations when tested against M. aurum, M. bovis BCG, M. tuberculosis H37Rv and multi-drug resistant strains of TB-clinical isolates. In addition, the synthetic compounds inhibited mycobacterial drug efflux mechanism as we report for the first time for this class of compounds. These studies suggest that synthesized methyldisulfides are novel chemical scaffolds, which potentially can lead to the design of new drugs against TB. The inhibition of efflux pumps by these compounds is promising as it would be a way to improve the efficacy and/or extend the clinical utility of existing antibiotics.
This article is an open access article distributed under the terms and conditions of the Creative... more This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY
Carvotacetones (1-7) isolated from Sphaeranthus africanus were screened for their antimycobacteri... more Carvotacetones (1-7) isolated from Sphaeranthus africanus were screened for their antimycobacterial and efflux pump (EP) inhibitory potential against the mycobacterial model strains Mycobacterium smegmatis mc 2 155, Mycobacterium aurum ATCC 23366, and Mycobacterium bovis BCG ATCC 35734. The minimum inhibitory concentrations (MICs) of the carvotacetones were detected through high-throughput spot culture growth inhibition (HT-SPOTi) and microbroth dilution assays. In order to assess the potential of the compounds 1 and 6 to accumulate ethidium bromide (EtBr) in M. smegmatis and M. aurum, a microtiter plate-based fluorometric assay was used to determine efflux activity. Compounds 1 and 6 were analyzed for their modulating effects on the MIC of EtBr and the antibiotic rifampicin (RIF) against M. smegmatis. Carvotacetones 1 and 6 had potent antibacterial effects on M. aurum and M. bovis BCG (MIC ≤ 31.25 mg/L) and could successfully enhance EtBr activity against M. smegmatis. Compound 1 appeared as the most efficient agent for impairing the efflux mechanism in M. smegmatis. Both compounds 1 and 6 were highly effective against M. aurum and M. bovis BCG. In particular, compound 1 was identified as a valuable candidate for inhibiting mycobacterial efflux mechanisms and as a promising adjuvant in the therapy of tuberculosis or other non-tubercular mycobacterial infections.
A series of imidazole and triazole diarylpyrazole derivatives were prepared using an efficient 5-... more A series of imidazole and triazole diarylpyrazole derivatives were prepared using an efficient 5-step synthetic scheme and evaluated for binding affinity with Mycobacterium tuberculosis (Mtb) CYP121A1 and antimycobacterial activity against Mtb H37Rv. Antimycobacterial susceptibility was measured using the spotculture growth inhibition assay (SPOTi): the imidazoles displayed minimum inhibitory concentration (MIC 90) in the range of 3.95-12.03 μg mL −1 (10.07-33.19 μM) with 11f the most active, while the triazoles displayed MIC 90 in the range of 4.35-25.63 μg mL −1 (11.88-70.53 μM) with 12b the most active. Assessment of binding affinity using UV-vis spectroscopy showed that for the imidazole series, the propyloxy (11f) and isopropyloxy (11h) derivatives of the 4-chloroaryl pyrazoles displayed Mtb CYP121A1 type II binding affinity with K d 11.73 and 17.72 μM respectively compared with the natural substrate cYY (K d 12.28 μM), while in the triazole series, only the methoxy substitution with the 4-chloroaryl pyrazole (12b) showed good type II Mtb CYP121A1 binding affinity (K d 5.13 μM). Protein-detected 1D 19 F-NMR spectroscopy as an orthogonal strategy was used to evaluate ligand binding independent of perturbations at the haem. For imidazole and triazole compounds, perturbations were more intense than cYY indicating tighter binding and confirming that ligand coordination occurs in the substrate-binding pocket despite very modest changes in UV-vis absorbance, consistent with computational studies and the demonstrated potential anti-tuberculosis properties of these compounds.
A nail patch is an attractive option for the topical treatment of onychomycosis, although no prod... more A nail patch is an attractive option for the topical treatment of onychomycosis, although no product is commercially available. We previously identified optimal nail patch formulations for two anti-onychomycotic drugs, based on their properties, as well as those of the other patch components. In this paper, our aim was to further investigate the potential of the patch formulations as topical nail medicines, in particular, whether the drug-in-adhesive patches release drug which then permeates into and through the nail plate and show anti-fungal efficacy, and whether and to what extent they remain adhered to the human nail plate in vivo when tested over 2 week durations. In addition, the influence of the drug (amorolfine HCl, ciclopirox olamine) and PSA (Duro-Tak 2852 or Duro-Tak 202A) on these parameters was determined. We found that both the nature of the drug and of the PSA influenced in vitro drug release. The nature of the drug, but not that of the PSA, influenced ungual drug permeation through human nail clippings, with considerably greater (almost double) permeation for ciclopirox olamine, the smaller and less lipophilic molecule. In vivo residence, tested with 3 out of the 4 patches, excluding the patch where ciclopirox olamine degraded with time, showed greater residence on toenails compared to fingernails reflecting their far lesser exposure to environmental stresses during daily activities. In vivo residence was enhanced when the patch was cut to the shape of the nail, was applied at bedtime, and when a clear colourless nail varnish was applied on top of the patch to 'seal' it into place on the nail. Comparison of the patches indicated greater residence of Duro-Tak 202A containing patches over those containing Duro-Tak 2852. Amorolfine HCl in Duro-Tak 202A based patch also showed antifungal efficacy in contrast to Duro-Tak 2852-based patch, and is particularly promising for further development as a potential toenail medicine, remaining almost fully adhered to toenails for at least two weeks.
The product of the gene ponA present in cosmid MTCY21D4, one of the collection of clones represen... more The product of the gene ponA present in cosmid MTCY21D4, one of the collection of clones representing the genome of Mycobacterium tuberculosis, has been named penicillin-binding protein 1* (PBP1*), by analogy to the previously characterized PBP1* of M. leprae. This gene has been overexpressed in Escherichia coli. His '-tagged PBP1* localizes to the membranes of induced E. coli cells. Its susceptibility to degradation upon proteinase K digestion of spheroplasts from E. coli expressing the protein supports the view that the majority of the protein translocates to the periplasmic side of the membrane. Recombinant PBP1* binds benzylpenicillin and several other β-lactams, notably cefotaxime, with high affinity. Truncation of the Nterminal 64 amino acid residues results in an expressed protein
The product of the gene ponA present in cosmid MTCY21D4, one of the collection of clones represen... more The product of the gene ponA present in cosmid MTCY21D4, one of the collection of clones representing the genome of Mycobacterium tuberculosis, has been named penicillin-binding protein 1* (PBP1*), by analogy to the previously characterized PBP1* of M. leprae. This gene has been overexpressed in Escherichia coli. His '-tagged PBP1* localizes to the membranes of induced E. coli cells. Its susceptibility to degradation upon proteinase K digestion of spheroplasts from E. coli expressing the protein supports the view that the majority of the protein translocates to the periplasmic side of the membrane. Recombinant PBP1* binds benzylpenicillin and several other β-lactams, notably cefotaxime, with high affinity. Truncation of the Nterminal 64 amino acid residues results in an expressed protein
Current trends on biotechnology & microbiology, Sep 6, 2019
Antimicrobial resistance (AMR) is a growing problem worldwide. Resistance to antibiotics can occu... more Antimicrobial resistance (AMR) is a growing problem worldwide. Resistance to antibiotics can occur in a number of ways, one of which is removal of the drugs from the cell via efflux pump macromolecular machineries. As such, efflux pumps can provide a background level of resistance to many different classes of antimicrobials and are a major contributor to AMR. Inhibition of efflux pumps therefore has the potential to reverse resistance to many antibiotics in one go and is an attractive potential for treating resistant infections. Whilst a number of efflux inhibitors are known, none are currently used clinically due to harmful side effects. Development of novel inhibitors is therefore imperative. The article aims to review accumulation assays and efflux assays, two of the most common laboratory techniques used to identify and characterise candidate efflux inhibitors.
Strains of members of Enterobacteriaceae, namely Escherichia coli (18), Klebsiella aerogenes (16)... more Strains of members of Enterobacteriaceae, namely Escherichia coli (18), Klebsiella aerogenes (16), and Serratia marcescens (16) were screened for Cd resistance or sensitivity. Only one strain each of these was resistant to high levels (25 n moles/0.05 ml) CdCl2. The Minimal inhibitory concentration (MIC) of sensitive strains ranged from 0.8-5 micrograms/ml. All the resistant strains were simultaneously resistant to a number of antibiotics. Treatment with sodium dodecyl sulfate eliminated resistance to Cd and to some antibiotics.
This article is an open access article distributed under the terms and conditions of the Creative... more This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY
This is a PDF file of an article that has undergone enhancements after acceptance, such as the ad... more This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
From Allium stipitatum, three pyridine-N-oxide alkaloids (1-3) possessing disulfide functional gr... more From Allium stipitatum, three pyridine-N-oxide alkaloids (1-3) possessing disulfide functional groups were isolated. The structures of these natural products were elucidated by spectroscopic means as 2-(methyldithio)pyridine-N-oxide (1), 2-[(methylthiomethyl)dithio]pyridine-N-oxide (2), and 2,2′-dithio-bis-pyridine-N-oxide (3). The proposed structure of 1 was confirmed by synthetic Smethylthiolation of commercial 2-thiopyridine-N-oxide. Compounds 1 and 2 are new natural products, and 3 is reported for the first time from an Allium species. All compounds were evaluated for activity against fast-growing species of Mycobacterium, methicillin-resistant Staphylococcus aureus, and a multidrug-resistant (MDR) variants of S. aureus. Compounds 1 and 2 exhibited minimum inhibitory concentrations (MICs) of 0.5-8 μg/mL against these strains. A small series of analogues of 1 were synthesized in an attempt to optimize antibacterial activity, although the natural product had the most potent in vitro activity. In a whole-cell assay at 30 μg/mL, 1 was shown to give complete inhibition of the incorporation of 14 C-labeled acetate into soluble fatty acids, indicating that it is potentially an inhibitor of fatty acid biosynthesis. In a human cancer cell line antiproliferative assay, 1 and 2 displayed IC 50 values ranging from 0.3 to 1.8 μM with a selectivity index of 2.3 when compared to a human somatic cell line. Compound 1 was evaluated in a microarray analysis that indicated a similar mode of action to menadione and 8-quinolinol by interfering with the thioredoxin system and up-regulating the production of various heat shock proteins. This compound was also assessed in a mouse model for in vivo toxicity. ¶ Dedicated to Dr. David G. I. Kingston of Virginia Polytechnic Institute and State University for his pioneering work on bioactive natural products.
This article is an open access article distributed under the terms and conditions of the Creative... more This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY
Journal of Infectious Diseases and Therapy, Sep 2, 2015
T uberculosis (TB), having been declared a global emergency since 1993, is still an issue of grea... more T uberculosis (TB), having been declared a global emergency since 1993, is still an issue of great public health concern because of the emergence of multi-and extensively-drug-resistant strains of Mycobacterium tuberculosis. New molecules with pleiotropic modes of action are urgently required to tackle this challenging menace. Pyridine-N-oxide alkaloids possessing disulfide functional groups were isolated from the bulbs of Allium stipitatum, belonging to the genus Allium with common members like garlic, onion, leeks and chives. From this discovery, a series of methyldisulfides were synthesized based on the structure of the natural product disulphides isolated from the bulbs of Allium stipitatum. The synthetic analogues were produced by adopting the method of Kitson and Loomes, briefly, the appropriate aromatic thiol purchased from Sigma Aldrich was S-methylthiolated using S-methyl methanethiosulphonate and back extracted with dichloromethane. Antibacterial activities using various high-throughput whole cell phenotypic assays were carried out. A selection of five compounds of that chemical class showed antimycobacterial activities at clinically relevant concentrations when tested against M. aurum, M. bovis BCG, M. tuberculosis H37Rv and multi-drug resistant strains of TB-clinical isolates. In addition, the synthetic compounds inhibited mycobacterial drug efflux mechanism as we report for the first time for this class of compounds. These studies suggest that synthesized methyldisulfides are novel chemical scaffolds, which potentially can lead to the design of new drugs against TB. The inhibition of efflux pumps by these compounds is promising as it would be a way to improve the efficacy and/or extend the clinical utility of existing antibiotics.
This article is an open access article distributed under the terms and conditions of the Creative... more This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY
Carvotacetones (1-7) isolated from Sphaeranthus africanus were screened for their antimycobacteri... more Carvotacetones (1-7) isolated from Sphaeranthus africanus were screened for their antimycobacterial and efflux pump (EP) inhibitory potential against the mycobacterial model strains Mycobacterium smegmatis mc 2 155, Mycobacterium aurum ATCC 23366, and Mycobacterium bovis BCG ATCC 35734. The minimum inhibitory concentrations (MICs) of the carvotacetones were detected through high-throughput spot culture growth inhibition (HT-SPOTi) and microbroth dilution assays. In order to assess the potential of the compounds 1 and 6 to accumulate ethidium bromide (EtBr) in M. smegmatis and M. aurum, a microtiter plate-based fluorometric assay was used to determine efflux activity. Compounds 1 and 6 were analyzed for their modulating effects on the MIC of EtBr and the antibiotic rifampicin (RIF) against M. smegmatis. Carvotacetones 1 and 6 had potent antibacterial effects on M. aurum and M. bovis BCG (MIC ≤ 31.25 mg/L) and could successfully enhance EtBr activity against M. smegmatis. Compound 1 appeared as the most efficient agent for impairing the efflux mechanism in M. smegmatis. Both compounds 1 and 6 were highly effective against M. aurum and M. bovis BCG. In particular, compound 1 was identified as a valuable candidate for inhibiting mycobacterial efflux mechanisms and as a promising adjuvant in the therapy of tuberculosis or other non-tubercular mycobacterial infections.
A series of imidazole and triazole diarylpyrazole derivatives were prepared using an efficient 5-... more A series of imidazole and triazole diarylpyrazole derivatives were prepared using an efficient 5-step synthetic scheme and evaluated for binding affinity with Mycobacterium tuberculosis (Mtb) CYP121A1 and antimycobacterial activity against Mtb H37Rv. Antimycobacterial susceptibility was measured using the spotculture growth inhibition assay (SPOTi): the imidazoles displayed minimum inhibitory concentration (MIC 90) in the range of 3.95-12.03 μg mL −1 (10.07-33.19 μM) with 11f the most active, while the triazoles displayed MIC 90 in the range of 4.35-25.63 μg mL −1 (11.88-70.53 μM) with 12b the most active. Assessment of binding affinity using UV-vis spectroscopy showed that for the imidazole series, the propyloxy (11f) and isopropyloxy (11h) derivatives of the 4-chloroaryl pyrazoles displayed Mtb CYP121A1 type II binding affinity with K d 11.73 and 17.72 μM respectively compared with the natural substrate cYY (K d 12.28 μM), while in the triazole series, only the methoxy substitution with the 4-chloroaryl pyrazole (12b) showed good type II Mtb CYP121A1 binding affinity (K d 5.13 μM). Protein-detected 1D 19 F-NMR spectroscopy as an orthogonal strategy was used to evaluate ligand binding independent of perturbations at the haem. For imidazole and triazole compounds, perturbations were more intense than cYY indicating tighter binding and confirming that ligand coordination occurs in the substrate-binding pocket despite very modest changes in UV-vis absorbance, consistent with computational studies and the demonstrated potential anti-tuberculosis properties of these compounds.
A nail patch is an attractive option for the topical treatment of onychomycosis, although no prod... more A nail patch is an attractive option for the topical treatment of onychomycosis, although no product is commercially available. We previously identified optimal nail patch formulations for two anti-onychomycotic drugs, based on their properties, as well as those of the other patch components. In this paper, our aim was to further investigate the potential of the patch formulations as topical nail medicines, in particular, whether the drug-in-adhesive patches release drug which then permeates into and through the nail plate and show anti-fungal efficacy, and whether and to what extent they remain adhered to the human nail plate in vivo when tested over 2 week durations. In addition, the influence of the drug (amorolfine HCl, ciclopirox olamine) and PSA (Duro-Tak 2852 or Duro-Tak 202A) on these parameters was determined. We found that both the nature of the drug and of the PSA influenced in vitro drug release. The nature of the drug, but not that of the PSA, influenced ungual drug permeation through human nail clippings, with considerably greater (almost double) permeation for ciclopirox olamine, the smaller and less lipophilic molecule. In vivo residence, tested with 3 out of the 4 patches, excluding the patch where ciclopirox olamine degraded with time, showed greater residence on toenails compared to fingernails reflecting their far lesser exposure to environmental stresses during daily activities. In vivo residence was enhanced when the patch was cut to the shape of the nail, was applied at bedtime, and when a clear colourless nail varnish was applied on top of the patch to 'seal' it into place on the nail. Comparison of the patches indicated greater residence of Duro-Tak 202A containing patches over those containing Duro-Tak 2852. Amorolfine HCl in Duro-Tak 202A based patch also showed antifungal efficacy in contrast to Duro-Tak 2852-based patch, and is particularly promising for further development as a potential toenail medicine, remaining almost fully adhered to toenails for at least two weeks.
The product of the gene ponA present in cosmid MTCY21D4, one of the collection of clones represen... more The product of the gene ponA present in cosmid MTCY21D4, one of the collection of clones representing the genome of Mycobacterium tuberculosis, has been named penicillin-binding protein 1* (PBP1*), by analogy to the previously characterized PBP1* of M. leprae. This gene has been overexpressed in Escherichia coli. His '-tagged PBP1* localizes to the membranes of induced E. coli cells. Its susceptibility to degradation upon proteinase K digestion of spheroplasts from E. coli expressing the protein supports the view that the majority of the protein translocates to the periplasmic side of the membrane. Recombinant PBP1* binds benzylpenicillin and several other β-lactams, notably cefotaxime, with high affinity. Truncation of the Nterminal 64 amino acid residues results in an expressed protein
Polymorphic Human arylamine N-acetyltransferase (NAT2) inactivates the anti-tubercular drug isoni... more Polymorphic Human arylamine N-acetyltransferase (NAT2) inactivates the anti-tubercular drug isoniazid by acetyltransfer from acetylCoA. There are active NAT proteins encoded by homologous genes in mycobacteria including M. tuberculosis, M. bovis BCG, M. smegmatis and M. marinum. Crystallographic structures of NATs from M. smegmatis and M. marinum, as native enzymes and with isoniazid bound share a similar fold with the first NAT structure, Salmonella typhimurium NAT. There are three approximately equal domains and an active site essential catalytic triad of cysteine, histidine and aspartate in the first two domains. An acetyl group from acetylCoA is transferred to cysteine and then to the acetyl acceptor e.g. isoniazid. M. marinum NAT binds CoA in a more open mode compared with CoA binding to human NAT2. The structure of mycobacterial NAT may promote its role in synthesis of cell wall lipids, identified through gene deletion studies. NAT protein is essential for survival of M. bovis BCG in macrophage as are the proteins encoded by other genes in the same gene cluster (hsaA-D). HsaA-D degrade cholesterol, essential for mycobacterial survival inside macrophage. Nat expression remains to be fully understood but is co-ordinated with hsaA-D and other stress response genes in mycobacteria. Amide synthase genes in the streptomyces are also nat homologues. The amide synthases are predicted to catalyse intramolecular amide bond formation and creation of cyclic molecules, e.g. geldanamycin. Lack of conservation of the CoA binding cleft residues of M. marinum NAT suggests the amide synthase reaction mechanism does not involve a soluble CoA intermediate during amide formation and ring closure.
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