cord-factors and arabinogalactan

The mycomembrane layer of the mycobacterial cell envelope is a barrier to environmental, immune, and antibiotic insults. There is considerable evidence of mycomembrane plasticity during infection and in response to host-mimicking stresses. Since mycobacteria are resource and energy limited under these conditions, it is likely that remodeling has distinct requirements from those of the well-characterized biosynthetic program that operates during unrestricted growth. Unexpectedly, we found that mycomembrane remodeling in nutrient-starved, nonreplicating mycobacteria includes synthesis in addition to turnover. Mycomembrane synthesis under these conditions occurs along the cell periphery, in contrast to the polar assembly of actively growing cells, and both liberates and relies on the nonmammalian disaccharide trehalose. In the absence of trehalose recycling,

Topics: Adenosine Triphosphate; Anti-Bacterial Agents; ATP-Binding Cassette Transporters; Bacterial Proteins; Cell Membrane; Cell Wall; Cord Factors; Diarylquinolines; Energy Metabolism; Galactans; Gene Expression; Glucose; Maltose; Membrane Transport Proteins; Mycobacterium smegmatis; Mycobacterium tuberculosis; Mycolic Acids; Rifampin; Trehalose

The

Topics: Antitubercular Agents; Bacterial Proteins; Benzamides; Benzothiazoles; Binding Sites; Biological Transport; Cord Factors; Drug Resistance, Bacterial; Galactans; Gene Expression; High-Throughput Screening Assays; Membrane Transport Proteins; Microbial Sensitivity Tests; Models, Molecular; Mutation; Mycobacterium abscessus; Mycobacterium tuberculosis; Mycolic Acids; Protein Binding; Protein Structure, Secondary; Pyridines; Recombinant Proteins; Whole Genome Sequencing

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Topics: Amino Acid Motifs; Antitubercular Agents; Bacterial Proteins; Benzimidazoles; Binding Sites; Biological Transport; Cloning, Molecular; Cord Factors; Drug Resistance, Bacterial; Escherichia coli; Galactans; Gene Expression; Genetic Vectors; Membrane Transport Proteins; Microbial Sensitivity Tests; Models, Molecular; Mutation; Mycobacterium tuberculosis; Mycolic Acids; Protein Binding; Protein Conformation, alpha-Helical; Recombinant Proteins; Whole Genome Sequencing

The three isoforms of antigen 85 (A, B, and C) are the most abundant secreted mycobacterial proteins and catalyze transesterification reactions that synthesize mycolated arabinogalactan, trehalose monomycolate (TMM), and trehalose dimycolate (TDM), important constituents of the outermost layer of the cellular envelope of Mycobacterium tuberculosis. These three enzymes are nearly identical at the active site and have therefore been postulated to exist to evade host immunity. Distal to the active site is a second putative carbohydrate-binding site of lower homology. Mutagenesis of the three isoforms at this second site affected both substrate selectivity and overall catalytic activity in vitro. Using synthetic and natural substrates, we show that these three enzymes exhibit unique selectivity; antigen 85A more efficiently mycolates TMM to form TDM, whereas C (and to a lesser extent B) has a higher rate of activity using free trehalose to form TMM. This difference in substrate selectivity extends to the hexasaccharide fragment of cell wall arabinan. Mutation of secondary site residues from the most active isoform (C) into those present in A or B partially interconverts this substrate selectivity. These experiments in combination with molecular dynamics simulations reveal that differences in the N-terminal helix α9, the adjacent Pro(216)-Phe(228) loop, and helix α5 are the likely cause of changes in activity and substrate selectivity. These differences explain the existence of three isoforms and will allow for future work in developing inhibitors.

Topics: Acyltransferases; Amino Acid Sequence; Antigens, Bacterial; Bacterial Proteins; Binding Sites; Biocatalysis; Carbohydrate Sequence; Catalytic Domain; Cell Wall; Cord Factors; Galactans; Molecular Dynamics Simulation; Molecular Sequence Data; Mutation; Mycobacterium tuberculosis; Polysaccharides; Protein Binding; Protein Structure, Secondary; Sequence Homology, Amino Acid; Substrate Specificity