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

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

Author information

1
From the Tuberculosis Research Section, Laboratory of Clinical Infectious Diseases, and the Department of Chemistry, University of Oxford, Chemistry Research Laboratory, Oxford OX1 3TA, United Kingdom.
2
the Bioinformatics and Computational Biosciences Branch, NIAID, National Institutes of Health, Bethesda, Maryland 20892.
3
the Department of Chemistry, University of Oxford, Chemistry Research Laboratory, Oxford OX1 3TA, United Kingdom.
4
the Alberta Glycomics Centre and Department of Chemistry, University of Alberta, Edmonton, Alberta T6G 2G2, Canada, and.
5
the Laboratory of Biochemistry and Genetics, NIDDK, National Institutes of Health, Bethesda, Maryland 20892.
6
From the Tuberculosis Research Section, Laboratory of Clinical Infectious Diseases, and.
7
the Department of Chemistry, University of Oxford, Chemistry Research Laboratory, Oxford OX1 3TA, United Kingdom, Ben.Davis@chem.ox.ac.uk.
8
From the Tuberculosis Research Section, Laboratory of Clinical Infectious Diseases, and cbarry@niaid.nih.gov.

Abstract

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

PMID:
25028517
PMCID:
PMC4155671
DOI:
10.1074/jbc.M114.581579
[Indexed for MEDLINE]
Free PMC Article

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