The three Mycobacterium tuberculosis antigen 85 isoforms have unique substrates and activities determined by non-active site regions

J Biol Chem. 2014 Sep 5;289(36):25041-53. doi: 10.1074/jbc.M114.581579. Epub 2014 Jul 14.


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.

Keywords: Antigen 85; Cell Wall; Enzyme Mechanism; Glycolipid; Molecular Dynamics; Mycolyl Transferase; Trehalose; Tuberculosis.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, N.I.H., Intramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Acyltransferases / chemistry
  • Acyltransferases / genetics
  • Acyltransferases / metabolism*
  • Amino Acid Sequence
  • Antigens, Bacterial / chemistry
  • Antigens, Bacterial / genetics
  • Antigens, Bacterial / metabolism*
  • Bacterial Proteins / chemistry
  • Bacterial Proteins / genetics
  • Bacterial Proteins / metabolism*
  • Binding Sites / genetics
  • Biocatalysis
  • Carbohydrate Sequence
  • Catalytic Domain
  • Cell Wall / enzymology
  • Cell Wall / metabolism
  • Cord Factors / metabolism
  • Galactans / metabolism
  • Molecular Dynamics Simulation
  • Molecular Sequence Data
  • Mutation
  • Mycobacterium tuberculosis / enzymology*
  • Mycobacterium tuberculosis / genetics
  • Mycobacterium tuberculosis / metabolism
  • Polysaccharides / metabolism
  • Protein Binding
  • Protein Structure, Secondary
  • Sequence Homology, Amino Acid
  • Substrate Specificity


  • Antigens, Bacterial
  • Bacterial Proteins
  • Cord Factors
  • Galactans
  • Polysaccharides
  • trehalose monomycolate
  • araban
  • Acyltransferases
  • antigen 85A, Mycobacterium tuberculosis
  • antigen 85B, Mycobacterium tuberculosis
  • antigen 85C, Mycobacterium tuberculosis
  • arabinogalactan