Abstract
Mycobacterium tuberculosis antigen 85 (Ag85) enzymes catalyze the transfer of mycolic acid (MA) from trehalose monomycolate to produce the mycolyl arabinogalactan (mAG) or trehalose dimycolate (TDM). These lipids define the protective mycomembrane of mycobacteria. The current model of substrate binding within the active sites of Ag85s for the production of TDM is not sterically and geometrically feasible; additionally, this model does not account for the production of mAG. Furthermore, this model does not address how Ag85s limit the hydrolysis of the acyl-enzyme intermediate while catalyzing acyl transfer. To inform an updated model, we obtained an Ag85 acyl-enzyme intermediate structure that resembles the mycolated form. Here, we present a 1.45-Å X-ray crystal structure of M. tuberculosis Ag85C covalently modified by tetrahydrolipstatin (THL), an esterase inhibitor that suppresses M. tuberculosis growth and mimics structural attributes of MAs. The mode of covalent inhibition differs from that observed in the reversible inhibition of the human fatty-acid synthase by THL. Similarities between the Ag85-THL structure and previously determined Ag85C structures suggest that the enzyme undergoes structural changes upon acylation, and positioning of the peptidyl arm of THL limits hydrolysis of the acyl-enzyme adduct. Molecular dynamics simulations of the modeled mycolated-enzyme form corroborate the structural analysis. From these findings, we propose an alternative arrangement of substrates that rectifies issues with the previous model and suggest a direct role for the -hydroxy of MA in the second half-reaction of Ag85 catalysis. This information affords the visualization of a complete mycolyltransferase catalytic cycle.
| Original language | English |
|---|---|
| Pages (from-to) | 3651-3662 |
| Number of pages | 12 |
| Journal | Journal of Biological Chemistry |
| Volume | 293 |
| Issue number | 10 |
| DOIs | |
| State | Published - Mar 9 2018 |
| Externally published | Yes |
Funding
Acknowledgments—This research used resources of the Advanced Photon Source, a United States Department of Energy (DOE) Office of Science User Facility operated for the DOE Office of Science by Argonne National Laboratory under Contract DE-AC02-06CH11357. This work used resources of the Compute and Data Environment for Science (CADES) at Oak Ridge National Laboratory, which is managed by UT–Battelle, LLC for the United States Department of Energy under Contract DE-AC05-00OR22725. This work was supported in part by National Institutes of Health Grant AI105084. This work has been co-authored by UT–Battelle, LLC under Con-tract DE-AC05-00OR22725 with the United States Department of Energy. The authors declare that they have no conflicts of interest with the con-tents of this article. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Insti-tutes of Health and the United States Department of Energy. This work was supported in part by National Institutes of Health Grant AI105084. This work has been co-authored by UT–Battelle, LLC under Contract DE-AC05-00OR22725 with the United States Department of Energy. The authors declare that they have no conflicts of interest with the contents of this article. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health and the United States Department of Energy.
| Funders | Funder number |
|---|---|
| Compute and Data Environment for Science | |
| DOE Office of Science | |
| Office of Science User Facility operated | |
| United States Department of Energy | |
| National Institutes of Health | |
| U.S. Department of Energy | |
| National Institute of Allergy and Infectious Diseases | R01AI105084 |
| U.S. Department of Labor | |
| Argonne National Laboratory | DE-AC02-06CH11357 |
| Oak Ridge National Laboratory | |
| University of Tampa | DE-AC05-00OR22725 |
