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Title: Mechanism-Based Inhibition of the Mycobacterium tuberculosis Branched-Chain Aminotransferase by d- and l-Cycloserine

Abstract

The branched-chain aminotransferase is a pyridoxal 5'-phosphate (PLP)-dependent enzyme responsible for the final step in the biosynthesis of all three branched-chain amino acids, l-leucine, l-isoleucine, and l-valine, in bacteria. We have investigated the mechanism of inactivation of the branched-chain aminotransferase from Mycobacterium tuberculosis (MtIlvE) by d- and l-cycloserine. d-Cycloserine is currently used only in the treatment of multidrug–drug-resistant tuberculosis. Our results show a time- and concentration-dependent inactivation of MtIlvE by both isomers, with l-cycloserine being a 40-fold better inhibitor of the enzyme. Minimum inhibitory concentration (MIC) studies revealed that l-cycloserine is a 10-fold better inhibitor of Mycobacterium tuberculosis growth than d-cycloserine. In addition, we have crystallized the MtIlvE-d-cycloserine inhibited enzyme, determining the structure to 1.7 Å. The structure of the covalent d-cycloserine-PMP adduct bound to MtIlvE reveals that the d-cycloserine ring is planar and aromatic, as previously observed for other enzyme systems. Mass spectrometry reveals that both the d-cycloserine- and l-cycloserine-PMP complexes have the same mass, and are likely to be the same aromatized, isoxazole product. However, the kinetics of formation of the MtIlvE d-cycloserine-PMP and MtIlvE l-cycloserine-PMP adducts are quite different. While the kinetics of the formation of the MtIlvE d-cycloserine-PMP complex can be fit to a single exponential,more » the formation of the MtIlvE l-cycloserine-PMP complex occurs in two steps. We propose a chemical mechanism for the inactivation of d- and l-cycloserine which suggests a stereochemically determined structural role for the differing kinetics of inactivation. Lastly, these results demonstrate that the mechanism of action of d-cycloserine’s activity against M. tuberculosis may be more complicated than previously thought and that d-cycloserine may compromise the in vivo activity of multiple PLP-dependent enzymes, including MtIlvE.« less

Authors:
 [1];  [1];  [1]; ORCiD logo [1]
  1. Albert Einstein College of Medicine, Bronx, NY (United States)
Publication Date:
Research Org.:
Argonne National Lab. (ANL), Argonne, IL (United States)
Sponsoring Org.:
USDOE Office of Science (SC); National Institutes of Health (NIH)
OSTI Identifier:
1506517
Grant/Contract Number:  
AC02-06CH11357
Resource Type:
Accepted Manuscript
Journal Name:
ACS Chemical Biology
Additional Journal Information:
Journal Volume: 12; Journal Issue: 5; Journal ID: ISSN 1554-8929
Publisher:
American Chemical Society (ACS)
Country of Publication:
United States
Language:
ENGLISH
Subject:
37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CHEMISTRY; 59 BASIC BIOLOGICAL SCIENCES; peptides and proteins; bacteria; genetics; monomers; chromatography

Citation Formats

Amorim Franco, Tathyana Mar, Favrot, Lorenza, Vergnolle, Olivia, and Blanchard, John S. Mechanism-Based Inhibition of the Mycobacterium tuberculosis Branched-Chain Aminotransferase by d- and l-Cycloserine. United States: N. p., 2017. Web. https://doi.org/10.1021/acschembio.7b00142.
Amorim Franco, Tathyana Mar, Favrot, Lorenza, Vergnolle, Olivia, & Blanchard, John S. Mechanism-Based Inhibition of the Mycobacterium tuberculosis Branched-Chain Aminotransferase by d- and l-Cycloserine. United States. https://doi.org/10.1021/acschembio.7b00142
Amorim Franco, Tathyana Mar, Favrot, Lorenza, Vergnolle, Olivia, and Blanchard, John S. Wed . "Mechanism-Based Inhibition of the Mycobacterium tuberculosis Branched-Chain Aminotransferase by d- and l-Cycloserine". United States. https://doi.org/10.1021/acschembio.7b00142. https://www.osti.gov/servlets/purl/1506517.
@article{osti_1506517,
title = {Mechanism-Based Inhibition of the Mycobacterium tuberculosis Branched-Chain Aminotransferase by d- and l-Cycloserine},
author = {Amorim Franco, Tathyana Mar and Favrot, Lorenza and Vergnolle, Olivia and Blanchard, John S.},
abstractNote = {The branched-chain aminotransferase is a pyridoxal 5'-phosphate (PLP)-dependent enzyme responsible for the final step in the biosynthesis of all three branched-chain amino acids, l-leucine, l-isoleucine, and l-valine, in bacteria. We have investigated the mechanism of inactivation of the branched-chain aminotransferase from Mycobacterium tuberculosis (MtIlvE) by d- and l-cycloserine. d-Cycloserine is currently used only in the treatment of multidrug–drug-resistant tuberculosis. Our results show a time- and concentration-dependent inactivation of MtIlvE by both isomers, with l-cycloserine being a 40-fold better inhibitor of the enzyme. Minimum inhibitory concentration (MIC) studies revealed that l-cycloserine is a 10-fold better inhibitor of Mycobacterium tuberculosis growth than d-cycloserine. In addition, we have crystallized the MtIlvE-d-cycloserine inhibited enzyme, determining the structure to 1.7 Å. The structure of the covalent d-cycloserine-PMP adduct bound to MtIlvE reveals that the d-cycloserine ring is planar and aromatic, as previously observed for other enzyme systems. Mass spectrometry reveals that both the d-cycloserine- and l-cycloserine-PMP complexes have the same mass, and are likely to be the same aromatized, isoxazole product. However, the kinetics of formation of the MtIlvE d-cycloserine-PMP and MtIlvE l-cycloserine-PMP adducts are quite different. While the kinetics of the formation of the MtIlvE d-cycloserine-PMP complex can be fit to a single exponential, the formation of the MtIlvE l-cycloserine-PMP complex occurs in two steps. We propose a chemical mechanism for the inactivation of d- and l-cycloserine which suggests a stereochemically determined structural role for the differing kinetics of inactivation. Lastly, these results demonstrate that the mechanism of action of d-cycloserine’s activity against M. tuberculosis may be more complicated than previously thought and that d-cycloserine may compromise the in vivo activity of multiple PLP-dependent enzymes, including MtIlvE.},
doi = {10.1021/acschembio.7b00142},
journal = {ACS Chemical Biology},
number = 5,
volume = 12,
place = {United States},
year = {2017},
month = {3}
}

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