title: The three Mycobacterium tuberculosis antigen 85 isoforms have unique substrates and activities determined by non-active site regions.
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.
authors.affiliation: 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.
authors.forename: Keriann M
authors.initials: KM
authors.lastname: Backus
authors.affiliation: the Bioinformatics and Computational Biosciences Branch, NIAID, National Institutes of Health, Bethesda, Maryland 20892.
authors.forename: Michael A
authors.initials: MA
authors.lastname: Dolan
authors.affiliation: the Department of Chemistry, University of Oxford, Chemistry Research Laboratory, Oxford OX1 3TA, United Kingdom.
authors.forename: Conor S
authors.initials: CS
authors.lastname: Barry
authors.affiliation: the Alberta Glycomics Centre and Department of Chemistry, University of Alberta, Edmonton, Alberta T6G 2G2, Canada, and.
authors.forename: Maju
authors.initials: M
authors.lastname: Joe
authors.affiliation: the Laboratory of Biochemistry and Genetics, NIDDK, National Institutes of Health, Bethesda, Maryland 20892.
authors.forename: Peter
authors.initials: P
authors.lastname: McPhie
authors.affiliation: From the Tuberculosis Research Section, Laboratory of Clinical Infectious Diseases, and.
authors.forename: Helena I M
authors.initials: HI
authors.lastname: Boshoff
authors.affiliation: the Alberta Glycomics Centre and Department of Chemistry, University of Alberta, Edmonton, Alberta T6G 2G2, Canada, and.
authors.forename: Todd L
authors.initials: TL
authors.lastname: Lowary
authors.affiliation: the Department of Chemistry, University of Oxford, Chemistry Research Laboratory, Oxford OX1 3TA, United Kingdom, Ben.Davis@chem.ox.ac.uk.
authors.forename: Benjamin G
authors.initials: BG
authors.lastname: Davis
authors.affiliation: From the Tuberculosis Research Section, Laboratory of Clinical Infectious Diseases, and cbarry@niaid.nih.gov.
authors.forename: Clifton E
authors.initials: CE
authors.lastname: Barry
mesh_terms: D000217:Acyltransferases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D000942:Antigens, Bacterial / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites / Q000235:genetics; D055162:Biocatalysis; D002240:Carbohydrate Sequence; D020134:Catalytic Domain; D002473:Cell Wall / Q000201:enzymology / Q000378:metabolism; D003311:Cord Factors / Q000378:metabolism; D005685:Galactans / Q000378:metabolism; D056004:Molecular Dynamics Simulation; D008969:Molecular Sequence Data; D009154:Mutation; D009169:Mycobacterium tuberculosis / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D011134:Polysaccharides / Q000378:metabolism; D011485:Protein Binding; D017433:Protein Structure, Secondary; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
chemical_list: D000942:Antigens, Bacterial; D001426:Bacterial Proteins; D003311:Cord Factors; D005685:Galactans; D011134:Polysaccharides; C021099:trehalose monomycolate; C030080:araban; D000217:Acyltransferases; C112580:antigen 85A, Mycobacterium tuberculosis; C121433:antigen 85B, Mycobacterium tuberculosis; C119774:antigen 85C, Mycobacterium tuberculosis; C005653:arabinogalactan
keywords: Antigen 85; Cell Wall; Enzyme Mechanism; Glycolipid; Molecular Dynamics; Mycolyl Transferase; Trehalose; Tuberculosis