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, 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. 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.
authors.affiliation: Department of Biochemistry, Albert Einstein College of Medicine , 1300 Morris Park Avenue, Bronx, New York 10461, United States.
authors.forename: Tathyana Mar
authors.initials: TM
authors.lastname: Amorim Franco
authors.affiliation: Department of Biochemistry, Albert Einstein College of Medicine , 1300 Morris Park Avenue, Bronx, New York 10461, United States.
authors.forename: Lorenza
authors.initials: L
authors.lastname: Favrot
authors.affiliation: Department of Biochemistry, Albert Einstein College of Medicine , 1300 Morris Park Avenue, Bronx, New York 10461, United States.
authors.forename: Olivia
authors.initials: O
authors.lastname: Vergnolle
authors.affiliation: Department of Biochemistry, Albert Einstein College of Medicine , 1300 Morris Park Avenue, Bronx, New York 10461, United States.
authors.forename: John S
authors.identifier: 0000-0002-9195-4402
authors.initials: JS
authors.lastname: Blanchard
mesh_terms: D018360:Crystallography, X-Ray; D003523:Cycloserine / Q000494:pharmacology*; D007700:Kinetics; D015394:Molecular Structure; D009169:Mycobacterium tuberculosis / Q000201:enzymology*; D013237:Stereoisomerism; D000637:Transaminases / Q000037:antagonists & inhibitors*
chemical_list: D003523:Cycloserine; D000637:Transaminases; C019798:branched-chain-amino-acid transaminase