Intercepting a Mycobacterial Biosynthetic Pathway with Covalent Labeling
Name
nihms-2148805.pdf
Description
Accepted version
Size
1.59 MB
Format
Adobe PDF
Checksum (MD5)
cc6a3399d742c9116625fd0cde558b4f
Author(s)
Warner, Theodore C
Marando, Victoria M
Santiago-Reyes, Omar A
Hart, Elizabeth M
Smelyansky, Stephanie R
Carter, Alan W
Bernhardt, Thomas G
Bryson, Bryan D
Kim, Daria E
Kiessling, Laura L
Date Issued
March 24, 2025
Journal
Journal of the American Chemical Society
Publisher
American Chemical Society
Citation
Theodore C. Warner, Victoria M. Marando, Omar A. Santiago-Reyes, Elizabeth M. Hart, Stephanie R. Smelyansky, Alan W. Carter, Thomas G. Bernhardt, Bryan D. Bryson, Daria E. Kim, Laura L. Kiessling; Intercepting a Mycobacterial Biosynthetic Pathway with Covalent Labeling. Journal American Chemical Society 2 April 2025; 147 (13): 11189–11198.
Version
Author's final manuscript
Abstract
The mycobacterial cell envelope plays both infectious and protective roles. Understanding its structure is crucial for unlocking the molecular basis underlying these functions. Studying glycans, the primary components of the cell envelope, is challenging due to their limited native functional handles for chemoselective modification. New labeling methods exploit biorthogonal chemistry, using small molecule mimics that intercept cellular metabolism or late-stage glycan biosynthesis. However, these strategies can have practical limitations, including probe delivery and effectiveness. An ideal small molecule probe should be easily deployed and exploit the critical enzyme–substrate relationships of natural substrates. To this end, we developed a “probegenic” strategy to label mycobacteria. Our approach eliminates the need for explicit substrate mimicry, as the relevant functionality is revealed by a target enzyme. Specifically, we synthesized an azide-substituted trans-β-lactone probe (AzLac), which adopts a substrate-like structure upon covalent enzyme labeling. This probe is incorporated by mycolyltransferases into a core mycobacterial cell envelope glycan, including in the pathogen Mycobacterium tuberculosis. Unlike other probes of the cell envelope, AzLac facilitates selective covalent labeling of the inner leaflet of the mycomembrane. Using Corynebacterium glutamicum mycolyltransferase deletion strains, we implicated Cmt2 as the primary mycolyltransferase target. We leveraged the ability to modify the cell envelope by demonstrating that AzLac could be used to attach a DNA barcode to mycobacteria, which would help track infection dynamics. Thus, we expect AzLac will be a valuable means of monitoring and tracking the mycobacterial cell envelope. Moreover, we anticipate masking and revealing recognition motifs in probes can be applied to diverse cellular targets.
MIT Department
Massachusetts Institute of Technology. Department of Chemistry
Terms of Use
Article is made available in accordance with the publisher's policy and may be subject to US copyright law. Please refer to the publisher's site for terms of use.
Persistent DSpace Link
DOI of Published Version
https://doi.org/10.1021/jacs.4c17913
