Mycobacterium tuberculosis CitA activity is modulated by cysteine oxidation and pyruvate binding.

As an adaptation for survival during infection, Mycobacterium tuberculosis becomes dormant, reducing its metabolism and growth. Two types of citrate synthases have been identified in Mycobacterium tuberculosis, GltA2 and CitA. Previous work shows that overexpression of CitA, the secondary citrate synthase, stimulates the growth of Mycobacterium tuberculosis under hypoxic conditions without showing accumulation of triacylglycerols and makes mycobacteria more sensitive to antibiotics, suggesting that CitA may play a role as a metabolic switch during infection and may be an interesting TB drug target. To assess the druggability and possible mechanisms of targeting CitA with small-molecule compounds, the CitA crystal structure was solved to 2.1 Å by X-ray crystallography. The solved structure shows that CitA lacks an NADH binding site that would afford allosteric regulation, which is atypical of most citrate synthases. However, a pyruvate molecule is observed within the analogous domain, suggesting pyruvate may instead be the allosteric regulator for CitA. The R149 and R153 residues forming the charged portion of the pyruvate binding pocket were mutated to glutamate and methionine, respectively, to assess the effect of mutations on activity. Protein thermal shift assay shows thermal stabilization of CitA in the presence of pyruvate compared to the two CitA variants designed to decrease pyruvate affinity. Solved crystal structures of both variants show no significant structural changes. However, the catalytic efficiency of the R153M variant increases by 2.6-fold. Additionally, we show that covalent modification of C143 of CitA by Ebselen completely arrests enzyme activity. Similar inhibition is observed using two spirocyclic Michael acceptor containing compounds, which inhibit CitA with ICapp50 values of 6.6 and 10.9 µM. A crystal structure of CitA modified by Ebselen was solved, but significant structural changes were lacking. Considering that covalent modification of C143 inactivates CitA and the proximity of C143 to the pyruvate binding site, this suggests that structural and/or chemical changes in this sub-domain are responsible for regulating CitA enzymatic activity.

The Mycobacterium tuberculosis mycothiol S-transferase is divalent metal-dependent for mycothiol binding and transfer.

Mycothiol S-transferase (MST) (encoded by the rv0443 gene) was previously identified as the enzyme responsible for the transfer of Mycothiol (MSH) to xenobiotic acceptors in Mycobacterium tuberculosis (M.tb) during xenobiotic stress. To further characterize the functionality of MST in vitro and the possible roles in vivo, X-ray crystallographic, metal-dependent enzyme kinetics, thermal denaturation studies, and antibiotic MIC determination in rv0433 knockout strain were performed. The binding of MSH and Zn2+ increases the melting temperature by 12.9 °C as a consequence of the cooperative stabilization of MST by both MSH and metal. The co-crystal structure of MST in complex with MSH and Zn2+ to 1.45 Å resolution supports the specific utilization of MSH as a substrate as well as affording insights into the structural requirements of MSH binding and the metal-assisted catalytic mechanism of MST. Contrary to the well-defined role of MSH in mycobacterial xenobiotic responses and the ability of MST to bind MSH, cell-based studies with an M.tb rv0443 knockout strain failed to provide evidence for a role of MST in processing of rifampicin or isoniazid. These studies suggest the necessity of a new direction to identify acceptors of the enzyme and better define the biological role of MST in mycobacteria.

Biochemical Characterization of the Mycobacterium smegmatis Threonine Deaminase.

The biosynthesis of branched-chain amino acids or BCAAs (l-isoleucine, l-leucine, and l-valine) is essential in eubacteria, but mammals are branched-chain amino acid auxotrophs, making the enzymes in the pathway excellent targets for antibacterial drug development. The biosynthesis of l-isoleucine, l-leucine, and l-valine is very efficient, requiring only eight enzymes. Threonine dehydratase (TD), a pyridoxal 5'-phosphate (PLP)-dependent enzyme encoded by the ilvA gene, is the enzyme responsible for the conversion of l-threonine (l-Thr) to α-ketobutyrate, ammonia, and water, which is the first step in the biosynthesis of l-isoleucine. We have cloned, expressed, and biochemically characterized the reaction catalyzed by Mycobacterium smegmatis TD (abbreviated as MsIlvA) using steady-state kinetics and kinetic isotope effects. We show here that in addition to l-threonine, l-allo-threonine and l-serine are also used as substrates by TD, and all exhibit sigmoidal, non-Michaelis-Menten kinetics. Curiously, ß-chloro-l-alanine was also a substrate rather than an inhibitor as expected. The enzymatic activity of TD is sensitive to the presence of allosteric regulators, including the activator l-valine or the end product feedback inhibitor of the BCAA pathway in which TD is involved, l-isoleucine. Primary deuterium kinetic isotopes are small, suggesting Cα proton abstraction is only partially rate-limiting. Solvent kinetic isotopes were significantly larger, indicating that a proton transfer occurring during the reaction is also partially rate-limiting. Finally, we demonstrate that l-cycloserine, a general inhibitor of PLP-dependent enzymes, is an excellent inhibitor of threonine deaminase.

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

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.

Post-translational Acetylation of MbtA Modulates Mycobacterial Siderophore Biosynthesis.

Iron is an essential element for life, but its soluble form is scarce in the environment and is rarer in the human body. Mtb (Mycobacterium tuberculosis) produces two aryl-capped siderophores, mycobactin (MBT) and carboxymycobactin (cMBT), to chelate intracellular iron. The adenylating enzyme MbtA catalyzes the first step of mycobactin biosynthesis in two half-reactions: activation of the salicylic acid as an acyl-adenylate and ligation onto the acyl carrier protein (ACP) domain of MbtB to form covalently salicylated MbtB-ACP. We report the first apo-MbtA structure from Mycobacterium smegmatis at 2.3 Å. We demonstrate here that MbtA activity can be reversibly, post-translationally regulated by acetylation. Indeed the mycobacterial Pat (protein lysine acetyltransferase), Rv0998, specifically acetylates MbtA on lysine 546, in a cAMP-dependent manner, leading to enzyme inhibition. MbtA acetylation can be reversed by the NAD+-dependent DAc (deacetyltransferase), Rv1151c. Deletion of Pat and DAc genes in Mtb revealed distinct phenotypes for strains lacking one or the other gene at low pH and limiting iron conditions. This study establishes a direct connection between the reversible acetylation system Pat/DAc and the ability of Mtb to adapt in limited iron conditions, which is critical for mycobacterial infection.

Bacterial GCN5-Related N-Acetyltransferases: From Resistance to Regulation.

The GCN5-related N-acetyltransferases family (GNAT) is an important family of proteins that includes more than 100000 members among eukaryotes and prokaryotes. Acetylation appears as a major regulatory post-translational modification and is as widespread as phosphorylation. N-Acetyltransferases transfer an acetyl group from acetyl-CoA to a large array of substrates, from small molecules such as aminoglycoside antibiotics to macromolecules. Acetylation of proteins can occur at two different positions, either at the amino-terminal end (αN-acetylation) or at the ε-amino group (εN-acetylation) of an internal lysine residue. GNAT members have been classified into different groups on the basis of their substrate specificity, and in spite of a very low primary sequence identity, GNAT proteins display a common and conserved fold. This Current Topic reviews the different classes of bacterial GNAT proteins, their functions, their structural characteristics, and their mechanism of action.

Inactivation of the Mycobacterium tuberculosis antigen 85 complex by covalent, allosteric inhibitors.

The rise of multidrug-resistant and totally drug-resistant tuberculosis and the association with an increasing number of HIV-positive patients developing tuberculosis emphasize the necessity to find new antitubercular targets and drugs. The antigen 85 (Ag85) complex from Mycobacterium tuberculosis plays important roles in the biosynthesis of major components of the mycobacterial cell envelope. For this reason, Ag85 has emerged as an attractive drug target. Recently, ebselen was identified as an effective inhibitor of the Ag85 complex through covalent modification of a cysteine residue proximal to the Ag85 active site and is therefore a covalent, allosteric inhibitor. To expand the understanding of this process, we have solved the x-ray crystal structures of Ag85C covalently modified with ebselen and other thiol-reactive compounds, p-chloromercuribenzoic acid and iodoacetamide, as well as the structure of a cysteine to glycine mutant. All four structures confirm that chemical modification or mutation at this particular cysteine residue leads to the disruption of the active site hydrogen-bonded network essential for Ag85 catalysis. We also describe x-ray crystal structures of Ag85C single mutants within the catalytic triad and show that a mutation of any one of these three residues promotes the same conformational change observed in the cysteine-modified forms. These results provide evidence for active site dynamics that may afford new strategies for the development of selective and potent Ag85 inhibitors.

Mechanism of inhibition of Mycobacterium tuberculosis antigen 85 by ebselen.

The increasing prevalence of drug-resistant tuberculosis highlights the need for identifying new antitubercular drugs that can treat these infections. The antigen 85 (Ag85) complex has emerged as an intriguing mycobacterial drug target due to its central role in synthesizing major components of the inner and outer leaflets of the mycobacterial outer membrane. Here we identify ebselen (EBS) as a potent inhibitor of the Mycobacterium tuberculosis Ag85 complex. Mass spectrometry data show that EBS binds covalently to a cysteine residue (C209) located near the Ag85C active site. The crystal structure of Ag85C in the presence of EBS shows that C209 modification restructures the active site, thereby disrupting the hydrogen-bonded network within the active site that is essential for enzymatic activity. C209 mutations display marked decreases in enzymatic activity. These data suggest that compounds using this mechanism of action will strongly inhibit the Ag85 complex and minimize the selection of drug resistance.

Targeting the mycobacterial envelope for tuberculosis drug development.

The bacterium that causes tuberculosis, Mycobacterium tuberculosis, possesses a rather unique outer membrane composed largely of lipids that possess long-chain and branched fatty acids, called mycolic acids. These lipids form a permeability barrier that prevents entry of many environmental solutes, thereby making these bacteria acid-fast and able to survive extremely hostile surroundings. Antitubercular drugs must penetrate this layer to reach their target. This review highlights drug development efforts that have added to the slowly growing tuberculosis drug pipeline, identified new enzyme activities to target with drugs and increased the understanding of important biosynthetic pathways for mycobacterial outer membrane and cell wall core assembly. In addition, a portion of this review looks at discovery efforts aimed at weakening this barrier to decrease mycobacterial virulence, decrease fitness in the host or enhance the efficacy of the current drug repertoire by disrupting the permeability barrier.