| Content Provider |
World Health Organization (WHO)-Global Index Medicus |
| Author |
Mcphie, Peter Backus, Keriann M. Dolan, Michael A. Barry, Conor S. Davis, Benjamin G. Joe, Maju Boshoff, Helena I. M. Lowary, Todd L. Barry, Clifton E. |
| Description |
Author Affiliation: Backus KM ( 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.); Dolan MA ( the Bioinformatics and Computational Biosciences Branch, NIAID, National Institutes of Health, Bethesda, Maryland 20892.); Barry CS ( the Department of Chemistry, University of Oxford, Chemistry Research Laboratory, Oxford OX1 3TA, United Kingdom.); Joe M ( the Alberta Glycomics Centre and Department of Chemistry, University of Alberta, Edmonton, Alberta T6G 2G2, Canada, and.); McPhie P ( the Laboratory of Biochemistry and Genetics, NIDDK, National Institutes of Health, Bethesda, Maryland 20892.); Boshoff HI ( From the Tuberculosis Research Section, Laboratory of Clinical Infectious Diseases, and.); Lowary TL ( the Alberta Glycomics Centre and Department of Chemistry, University of Alberta, Edmonton, Alberta T6G 2G2, Canada, and.); Davis BG ( the Department of Chemistry, University of Oxford, Chemistry Research Laboratory, Oxford OX1 3TA, United Kingdom, Ben.Davis@chem.ox.ac.uk.); Barry CE ( 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 9, the adjacent Pro(216)-Phe(228) loop, and helix 5 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. |
| ISSN |
00219258 |
| e-ISSN |
1083351X |
| Journal |
Journal of Biological Chemistry |
| Issue Number |
36 |
| Volume Number |
289 |
| Language |
English |
| Publisher |
American Society for Biochemistry and Molecular Biology (United States) |
| Publisher Date |
2014-09-05 |
| Publisher Place |
United States |
| Access Restriction |
Open |
| Subject Keyword |
Acyltransferases Metabolism Antigens, Bacterial Bacterial Proteins Mycobacterium Tuberculosis Enzymology Chemistry Genetics Amino Acid Sequence Binding Sites Biocatalysis Carbohydrate Sequence Catalytic Domain Cell Wall Cord Factors Galactans Molecular Dynamics Simulation Molecular Sequence Data Mutation Polysaccharides Protein Binding Protein Structure, Secondary Sequence Homology, Amino Acid Substrate Specificity Research Support, N.I.H., Extramural Research Support, N.I.H., Intramural Research Support, Non-U.S. Gov't Biochemistry Molecular Biology |
| Content Type |
Text |
| Resource Type |
Article |
| Subject |
Cell Biology Biochemistry Molecular Biology |
