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.

Intracellular Mycobacterium tuberculosis Exploits Multiple Host Nitrogen Sources during Growth in Human Macrophages

“…However, although our results confirm that Asp is indeed a nitrogen source, they also demonstrate that, of the tested amino acids, Gln is the principal source of nitrogen for intracellular Mtb and is the amino acid whose nitrogen is most widely assimilated (Figure 1C). Gln is the major fuel for a variety of mammalian cells including macrophages (Zhang et al., 2017), and THP-1 macrophages have previously been shown to assimilate Gln into Glu (Amorim Franco et al., 2017, Zhao et al., 2013, Choi and Park, 2018). Our previous study demonstrated that carbon from both Glu and Gln was available to Mtb inside the macrophage (Beste et al., 2013).…”

Bacterial Branched-Chain Amino Acid Biosynthesis: Structures, Mechanisms, and Drugability

“…137 This reaction is reversible and depends on the coenzyme PLP being covalently bound to the enzyme through a Schiff base with a protein lysine residue. 138 The first three-dimensional structure of a bacterial BCAT was reported in 1997.…”

“…A similar feature was observed when Mt IlvE was inhibited by d - and l -cycloserine. 137 d -Cycloserine is a second-line drug used in the treatment of MDR-TB and inhibits two sequential reactions of the peptidoglycan biosynthetic pathway: the PLP-dependent alanine racemase and d -alanine- d -alanine ligase. 149 Mt IlvE is inhibited by both cycloserine isomers in a time- and concentration-dependent fashion.…”

“…It was suggested that the cycloserine isomers, as well as the aminooxy compounds, are generalized inhibitors of PLP-dependent enzymes. 137 The mechanism of inactivation of Mt IlvE by the cycloserine isomers was shown to be the result of the aromatization of the cycloserine ring to form a stable PLP adduct. 137 The structure of the inhibited Mt IlvE– d -cycloserine complex was determined at 1.7 Å resolution and reveals an intact and planar d -cycloserine ring (Figure 8).…”

Recent advances for identification of new scaffolds and drug targets for Mycobacterium tuberculosis

“…Alanine racemase, an enzyme that performs racemization of l ‐alanine to d ‐alanine is involved in peptidoglycan biosynthesis. d ‐Cycloserine, a second line TB drug inhibits alanine racemase, d ‐alanine ligase, and l ‐alanine dehydrogenase enzymes from M. tuberculosis .…”