Gene_locus Report for: myctu-metx

Mycobacterium tuberculosis (Mtb) lacking functional homoserine transacetylase (HTA) is compromised in methionine biosynthesis, protein synthesis, and in the activity of multiple essential S-adenosyl-l-methionine-dependent enzymes. Additionally, deficient mutants are further disarmed by the toxic accumulation of lysine due to a redirection of the metabolic flux toward the lysine biosynthetic pathway. Studies with deletion mutants and crystallographic studies of the apoenzyme have, respectively, validated Mtb HTA as an essential enzyme and revealed a ligandable binding site. Seeking a mechanistic characterization of this enzyme, we report crucial structural details and comprehensive functional characterization of Mtb HTA. Crystallographic and mass spectral observation of the acetylated HTA intermediate and initial velocity studies were consistent with a ping-pong kinetic mechanism. Wild-type HTA and its site-directed mutants were kinetically characterized with a panel of natural and alternative substrates to understand substrate specificity and identify critical residues for catalysis. Titration experiments using fluorescence quenching showed that both substratesacetyl-CoA and l-homoserineengage in a strong and weak binding interaction with HTA. Additionally, substrate inhibition by acetyl-CoA and product inhibition by CoA and O-acetyl-l-homoserine were proposed to form the basis of a feedback regulation mechanism. By furnishing key mechanistic and structural information, these studies provide a foundation for structure-based design efforts around this attractive Mtb target.

The metA (Rv3341) gene from Mycobacterium tuberculosis H37Rv strain encodes a homoserine-acetyltransferase (HAT) enzyme, also called MetA. This enzyme plays a key role in the biosynthetic pathway of methionine and is a potential target for the development of antimicrobial drugs. Purified MetA showed 40 kDa molecular mass on SDS-PAGE. Manual docking was performed with substrates acetyl-CoA, l-homoserine, and p-nitrophenylacetate using crystal structure coordinates of MetA (PDB ID 6PUX) from M. tuberculosis. Multiple sequence alignment indicated that catalytic triad residues Ser157, Asp320, His350 were conserved across species in acetyltransferases, esterases, and hydrolases. As a conserved pentapeptide, GXSMG belongs to alpha/beta hydrolase superfamily and it shares similarity with esterases and hydrolases from different sources. Hydrolase activity of MetA was tested using (PNPA), N-acetylglycine, N-acetylmethionine and Phe-Gly as substrate. LC-MS confirmed that MetA possessed HAT activity, but no homoserine-succinyltransferase (HST) and serine-acetyltransferase (SAT) activities. Replacing acetyl-CoA with PNPA as acetyl group donor showed a drastic reduction in transferase activity, arising due to the interaction of R227 of the enzyme with PNPA. This could prevent the binding of the second substrate in the right orientation and results in the preferential transfer of the acetyl group to water, thus exhibiting hydrolase rather than transferase activity. In this paper, we report that MetA has both transferase and hydrolase activity depending on the correct orientation of the second substrate and the availability of the amino acids involved in enzyme-substrate interaction.

Mycobacterium tuberculosis is the cause of the world's most deadly infectious disease. Efforts are underway to target the methionine biosynthesis pathway, as it is not part of the host metabolism. The homoserine transacetylase MetX converts L-homoserine to O-acetyl-L-homoserine at the committed step of this pathway. In order to facilitate structure-based drug design, we determined the high-resolution crystal structures of three MetX proteins, including M. tuberculosis (MtMetX), Mycolicibacterium abscessus (MaMetX), and Mycolicibacterium hassiacum (MhMetX). A comparison of homoserine transacetylases from other bacterial and fungal species reveals a high degree of structural conservation amongst the enzymes. Utilizing homologous structures with bound cofactors, we analyzed the potential ligandability of MetX. The deep active-site tunnel surrounding the catalytic serine yielded many consensus clusters during mapping, suggesting that MtMetX is highly druggable.

Mycobacterium tuberculosis (Mtb) lacking functional homoserine transacetylase (HTA) is compromised in methionine biosynthesis, protein synthesis, and in the activity of multiple essential S-adenosyl-l-methionine-dependent enzymes. Additionally, deficient mutants are further disarmed by the toxic accumulation of lysine due to a redirection of the metabolic flux toward the lysine biosynthetic pathway. Studies with deletion mutants and crystallographic studies of the apoenzyme have, respectively, validated Mtb HTA as an essential enzyme and revealed a ligandable binding site. Seeking a mechanistic characterization of this enzyme, we report crucial structural details and comprehensive functional characterization of Mtb HTA. Crystallographic and mass spectral observation of the acetylated HTA intermediate and initial velocity studies were consistent with a ping-pong kinetic mechanism. Wild-type HTA and its site-directed mutants were kinetically characterized with a panel of natural and alternative substrates to understand substrate specificity and identify critical residues for catalysis. Titration experiments using fluorescence quenching showed that both substratesacetyl-CoA and l-homoserineengage in a strong and weak binding interaction with HTA. Additionally, substrate inhibition by acetyl-CoA and product inhibition by CoA and O-acetyl-l-homoserine were proposed to form the basis of a feedback regulation mechanism. By furnishing key mechanistic and structural information, these studies provide a foundation for structure-based design efforts around this attractive Mtb target.

The metA (Rv3341) gene from Mycobacterium tuberculosis H37Rv strain encodes a homoserine-acetyltransferase (HAT) enzyme, also called MetA. This enzyme plays a key role in the biosynthetic pathway of methionine and is a potential target for the development of antimicrobial drugs. Purified MetA showed 40 kDa molecular mass on SDS-PAGE. Manual docking was performed with substrates acetyl-CoA, l-homoserine, and p-nitrophenylacetate using crystal structure coordinates of MetA (PDB ID 6PUX) from M. tuberculosis. Multiple sequence alignment indicated that catalytic triad residues Ser157, Asp320, His350 were conserved across species in acetyltransferases, esterases, and hydrolases. As a conserved pentapeptide, GXSMG belongs to alpha/beta hydrolase superfamily and it shares similarity with esterases and hydrolases from different sources. Hydrolase activity of MetA was tested using (PNPA), N-acetylglycine, N-acetylmethionine and Phe-Gly as substrate. LC-MS confirmed that MetA possessed HAT activity, but no homoserine-succinyltransferase (HST) and serine-acetyltransferase (SAT) activities. Replacing acetyl-CoA with PNPA as acetyl group donor showed a drastic reduction in transferase activity, arising due to the interaction of R227 of the enzyme with PNPA. This could prevent the binding of the second substrate in the right orientation and results in the preferential transfer of the acetyl group to water, thus exhibiting hydrolase rather than transferase activity. In this paper, we report that MetA has both transferase and hydrolase activity depending on the correct orientation of the second substrate and the availability of the amino acids involved in enzyme-substrate interaction.

Mycobacterium tuberculosis is the cause of the world's most deadly infectious disease. Efforts are underway to target the methionine biosynthesis pathway, as it is not part of the host metabolism. The homoserine transacetylase MetX converts L-homoserine to O-acetyl-L-homoserine at the committed step of this pathway. In order to facilitate structure-based drug design, we determined the high-resolution crystal structures of three MetX proteins, including M. tuberculosis (MtMetX), Mycolicibacterium abscessus (MaMetX), and Mycolicibacterium hassiacum (MhMetX). A comparison of homoserine transacetylases from other bacterial and fungal species reveals a high degree of structural conservation amongst the enzymes. Utilizing homologous structures with bound cofactors, we analyzed the potential ligandability of MetX. The deep active-site tunnel surrounding the catalytic serine yielded many consensus clusters during mapping, suggesting that MtMetX is highly druggable.

The alpha/beta hydrolase fold superfamily is an ancient and widely diversified group of primarily hydrolytic enzymes. In this review, the adaptations of these proteins to the pathogenic lifestyle of Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis, are examined. Of the 105 alpha/beta hydrolases identified in Mtb, many are associated with lipid metabolism, particularly in the biosynthesis and maintenance of the Mtb's unique cell envelope, as well in the large number of extracellular lipases that are likely responsible for degradation of host lipid material. alpha/beta hydrolase fold proteins are also involved in the evasion and modulation of the immune response, detoxification and metabolic adaptations, including growth, response to acidification of the intracellular environment and dormancy. A striking feature of Mtb's alpha/beta hydrolases is their diversification into virulence-associated niches. It is clear that the alpha/beta hydrolase fold family has made a significant contribution to Mtb's remarkable success as a pathogen.

To investigate the molecular characteristics of bacillus Calmette-Guerin (BCG) vaccines, the complete genomic sequence of Mycobacterium bovis BCG Tokyo 172 was determined, and the results were compared with those for BCG Pasteur and other M. tuberculosis complex. The genome of BCG Tokyo had a length of 4,371,711bp and contained 4033 genes, including 3950 genes coding for proteins (CDS). There were 18 regions of difference (showing differences of more than 20bp), 20 insertion or deletion (ins/del) mutations of less than 20bp, and 68 SNPs between the two BCG substrains. These findings are useful for better understanding of the genetic differences in BCG substrains due to in vitro evolution of BCG.

To understand the evolution, attenuation, and variable protective efficacy of bacillus Calmette-Guerin (BCG) vaccines, Mycobacterium bovis BCG Pasteur 1173P2 has been subjected to comparative genome and transcriptome analysis. The 4,374,522-bp genome contains 3,954 protein-coding genes, 58 of which are present in two copies as a result of two independent tandem duplications, DU1 and DU2. DU1 is restricted to BCG Pasteur, although four forms of DU2 exist; DU2-I is confined to early BCG vaccines, like BCG Japan, whereas DU2-III and DU2-IV occur in the late vaccines. The glycerol-3-phosphate dehydrogenase gene, glpD2, is one of only three genes common to all four DU2 variants, implying that BCG requires higher levels of this enzyme to grow on glycerol. Further amplification of the DU2 region is ongoing, even within vaccine preparations used to immunize humans. An evolutionary scheme for BCG vaccines was established by analyzing DU2 and other markers. Lesions in genes encoding sigma-factors and pleiotropic transcriptional regulators, like PhoR and Crp, were also uncovered in various BCG strains; together with gene amplification, these affect gene expression levels, immunogenicity, and, possibly, protection against tuberculosis. Furthermore, the combined findings suggest that early BCG vaccines may even be superior to the later ones that are more widely used.

Mycobacterium bovis is the causative agent of tuberculosis in a range of animal species and man, with worldwide annual losses to agriculture of $3 billion. The human burden of tuberculosis caused by the bovine tubercle bacillus is still largely unknown. M. bovis was also the progenitor for the M. bovis bacillus Calmette-Guerin vaccine strain, the most widely used human vaccine. Here we describe the 4,345,492-bp genome sequence of M. bovis AF2122/97 and its comparison with the genomes of Mycobacterium tuberculosis and Mycobacterium leprae. Strikingly, the genome sequence of M. bovis is >99.95% identical to that of M. tuberculosis, but deletion of genetic information has led to a reduced genome size. Comparison with M. leprae reveals a number of common gene losses, suggesting the removal of functional redundancy. Cell wall components and secreted proteins show the greatest variation, indicating their potential role in host-bacillus interactions or immune evasion. Furthermore, there are no genes unique to M. bovis, implying that differential gene expression may be the key to the host tropisms of human and bovine bacilli. The genome sequence therefore offers major insight on the evolution, host preference, and pathobiology of M. bovis.

Virulence and immunity are poorly understood in Mycobacterium tuberculosis. We sequenced the complete genome of the M. tuberculosis clinical strain CDC1551 and performed a whole-genome comparison with the laboratory strain H37Rv in order to identify polymorphic sequences with potential relevance to disease pathogenesis, immunity, and evolution. We found large-sequence and single-nucleotide polymorphisms in numerous genes. Polymorphic loci included a phospholipase C, a membrane lipoprotein, members of an adenylate cyclase gene family, and members of the PE/PPE gene family, some of which have been implicated in virulence or the host immune response. Several gene families, including the PE/PPE gene family, also had significantly higher synonymous and nonsynonymous substitution frequencies compared to the genome as a whole. We tested a large sample of M. tuberculosis clinical isolates for a subset of the large-sequence and single-nucleotide polymorphisms and found widespread genetic variability at many of these loci. We performed phylogenetic and epidemiological analysis to investigate the evolutionary relationships among isolates and the origins of specific polymorphic loci. A number of these polymorphisms appear to have occurred multiple times as independent events, suggesting that these changes may be under selective pressure. Together, these results demonstrate that polymorphisms among M. tuberculosis strains are more extensive than initially anticipated, and genetic variation may have an important role in disease pathogenesis and immunity.

Countless millions of people have died from tuberculosis, a chronic infectious disease caused by the tubercle bacillus. The complete genome sequence of the best-characterized strain of Mycobacterium tuberculosis, H37Rv, has been determined and analysed in order to improve our understanding of the biology of this slow-growing pathogen and to help the conception of new prophylactic and therapeutic interventions. The genome comprises 4,411,529 base pairs, contains around 4,000 genes, and has a very high guanine + cytosine content that is reflected in the biased amino-acid content of the proteins. M. tuberculosis differs radically from other bacteria in that a very large portion of its coding capacity is devoted to the production of enzymes involved in lipogenesis and lipolysis, and to two new families of glycine-rich proteins with a repetitive structure that may represent a source of antigenic variation.