**3. The shunt and the hunt**

222 Understanding Tuberculosis – Deciphering the Secret Life of the Bacilli

by-products of its metabolism via the methylcitrate cycle are vital due to their potentially toxic effects but no homolog of this enzyme has been found in the mycobacterium genome. This unique phenomenon points out on the dual role of isocitrate lyase in the glyoxylate and methylcitrate cycles in *M. tuberculosis* (Gould et al., 2006). ICL-deficient bacteria could not grow on propionate, suggesting that ICL might function as ICLs in the glyoxylate cycle and

The enzyme malate synthase (EC 4.1.3.2) catalyses the condensation reaction between the carbonyl group of glyoxylate and the methyl group of acetyl-CoA to form a thio-ester

Catalysis by ICL and MS ensures the bypass of two oxidative steps of the tricarboxylic acid cycle, permitting net incorporation of carbon during growth of most microorganisms on acetate or fatty acids as the primary carbon source. Thus, the glyoxylate bypass conserves carbon and ensures an adequate supply of tricarboxylic acid cycle intermediates for biosynthetic purposes when cells convert lipids to carbohydrates

Magnesium serves as key ingredient in the recipe of lipid lunch as both the pivotal enzymes are activated in the presence of magnesium as a cofactor. There have been two schools of thoughts as far as role of magnesium on these enzymes are concerned the one thinks that magnesium is activating substrate by binding to it (magnesium-substrate complex) while other view rests on the idea that magnesium binds to the active site inducing conformational

The plant and bacterial ICL have an absolute requirement of Mg2+ ions for functional activity. Mg2+ ions were postulated to be necessary for catalysis on the active site of ICL however, a higher concentration of these ions has been found to have inhibitory effect on the enzyme (Beeckmans et al., 1997). Later on, Giachetti et al. (Giachetti & Vanni, 1991) performed detailed kinetic studies with *Pinus pinea* ICL and concluded that the Mg2+ isocitrate complex and not isocitrate is the true substrate of enzyme. This conclusion has been supported by studies on ICL from several other sources. In the absence of divalent cations, only negligible activity was measured for the purified ICL, whereas addition of Mg2+ or Mn2+ supported enzyme activity. Mn2+ was able to replace Mg2+, yielding 39% of the activity obtained with Mg2+. Co2+, Fe2+, Ca2+, Ba2+, Ni2+, Cd2+, Zn2+, Cu2+, and Hg2+ were not able to support significant ICL activity (Honer Zu Bentrup et al., 1999). A variety of metal ion combinations were studied for their ability to inhibit ICL activity. It is known for the isocitrate lyases of *Corynebacterium glutamicum* and *Acinetobacter calcoaceticus* that Mn2+ can partially substitute for Mg2+ (Hoyt et al., 1988; Hoyt et al., 1991).In the absence of divalent cations only negligible activity was measured for the purified MS. Mg2+ at 5 mM was found to be the most effective cation. Mn2+ was able to replace Mg2+, yielding 40% of the activity obtained with Mg2+, Co2+, Fe2+, Ca2+, Ba2+, Ni2+, Cd2+, Zn2+, Cu2+, and Hg2+ were not able to support significant MS activity (Smith et al., 2003). Zn2+ and Cd2+ were found to bring about structural alterations thereby inhibiting the function of enzyme in case of MtbIcl

as MCLs in the methylcitrate cycle (Munoz-Elias et al., 2006).

**2.3 Magnesium the magic molecule in metabolic recession** 

change and having catalytic role for efficient catalysis (2007).

(Sharma et al., 2000).

(Kumar & Bhakuni, 2008).

which, after hydrolysis, generates L-malate and CoA (Dixon et al., 1960).

The structural intricacies have been deciphered for both the key enzymes successfully and all the information is at our disposal (Sharma et al., 2000; Smith et al., 2003; Anstrom & Remington, 2006). In the era of structure based drug design where high throughput screening, molecular modelling, *in silico* docking strategies have accelerated drug development timeline promising rescue of the hijacked host from the persistent mycobacteria. The TB Alliance has been strategically focusing to combat these molecular targets. ICL is a tetrameric protein with four subunits of 428 amino acids each. The highresolution structure of ICL from *M. tuberculosis* has been solved to 2.0 Å resolution (Sharma et al., 2000). The enzyme structure in complex with inhibitors, 3-nitropropionate with glyoxylate and 3-bromopyruvate has also been resolved. 3-bromopyruvate inhibits ICLactivity by forming a covalent adduct with the nucleophilic Cys191 (Sharma et al., 2000). The inhibitor bound ICL structures, on one hand, provide crucial information regarding the active site microenvironment, and on the other hand, produce valuable information on the type of interactions prevalent at those localized site adding momentum strength to the drug discovery process. Several ICL inhibitors are being tested, which mainly include 3 nitropropionate (McFadden & Purohit, 1977), 3-bromopyruvate (Ko & McFadden, 1990), 3 phosphoglycerate (Ko et al., 1989), mycenon (Hautzel et al., 1990) and itaconate (McFadden & Purohit, 1977). However, *in vivo* application of these inhibitors is yet a dream because of their potent toxicity and low activity. Sesterterpene sulphate, which has recently been shown to effectively inhibit ICL in *Candida albicans* (Lee et al., 2008) is also promising.

The second enzyme of the glyoxylate shunt is encoded by a single gene identified in TB called *glcB* and encoding a 741 amino acid 80 kDa protein malate synthase (Smith et al., 2003). The enzyme catalyzes the Mg2+-dependent condensation of glyoxylate and acetylcoenzyme A and hydrolysis of the intermediate to yield malate and coenzyme A (Anstrom & Remington, 2006). The structure of MS from *M. tuberculosis* in complex with the substrate glyoxylate has been solved to 2.1 Å resolution structural analysis indicated that malate synthase is a much more druggable target by virtue of its deeper and more hydrophobic binding domain (Smith et al., 2003). Screening against this target will have a better chance of identifying tractable inhibitors as lead molecules (www.tballiance.org). Further refinement in understanding the mechanistic implications were brought forth by revised position of bound malate which is consistent with a reaction mechanism that does not require reorientation of the electrophilic substrate during the catalytic cycle(Anstrom & Remington, 2006) . These insights have been crucial in the inhibitor ergonomics. High throughput screening has been completed with a 1.4 million compound library and hits have been identified. The endeavour ahead is to confirm the potential hits and efficiently evaluate these, thus paving the pathway for identification of analogues and series for future optimization. It's also important to mention that high throughput screening initiatives for identifying inhibitors has not yielded very promising outcomes reason being the druggability of these potential targets. The challenge here is to design inhibitors that are equipped with permeability parameters that can reach the action targets.

Evolutionarily enzymes of glyoxylate shunt are highly conserved and have unique signature active site sequences which offer leverage to rational drug design approach thereby coming up with a broad spectrum more pharmacologically attractive target relevant to the treatment

*Mycobacterium tuberculosis*: Dormancy, Persistence and Survival in the Light of Protein Synthesis 225

process of the cells. The mechanism of translation and the function of the ribosome in mycobacteria can thus be visited collectively in the light of action of the drugs which include aminoglycosides, macrolides, tetracycline and peptide antibiotics etc. (Inderlied, 1991). At the same time the study of the resistance mechanism in mycobacteria against these drugs, often involving certain mutations on rRNA or r-proteins, would shed light on the process. In a study where several independent streptomycin-resistant mutants of *M. tuberculosis*  H37Rv were isolated, it was shown that while in the wild-type streptomycin inhibited the incorporation of 14C-amino acids into proteins, very little or no inhibition was observed in either high-level or low-level streptomycin-resistant strains (Shaila et al., 1973). This result on one hand points towards the activity of the drug against mycobacteria and on the other hand brings forward the potential problems arising from the development of resistance phenotypes. The Bottger-group has made very important contribution in the field in identifying potential drug candidates against mycobacteria (Hobbie et al., 2005; Hobbie et al., 2006a; Hobbie et al., 2006b; Hobbie et al., 2007). In addition, they have also identified potential hotspots for mutations leading to resistance against these drugs. Thus, these studies all together call for a continuous quest for identification of new and potential drug

Protein synthesis involves a coordinated action of the ribosome, mRNA, tRNAs and translation factors; thus it needs an intelligent setup and precise orchestration between different components. The process, also known as translation, is divided into four distinct steps; namely initiation, elongation, termination and ribosome-recycling. The process initiates by association of large ribosomal subunit with an mRNA programmed small subunit bound with initiator tRNA and initiation factors. Once a 70S initiation complex is successfully formed, the initiation factors dissociate and the process proceeds to elongation. In the first step of elongation the elongation factor–Tu (EF-Tu) brings tRNA loaded with respective amino acids to the decoding center (DC) of the ribosome. This site includes highly conserved regions of 16S rRNA arranged in a highly ordered structure. Correct codon anticodon pairing between the tRNA and mRNA through initial selection and tRNA accommodation leads the process towards the most significant step of protein synthesis, i.e. peptide bond formation. A primarily rRNA (23S rRNA) constituted region of the large subunit, called the Peptidyl-transferase site, catalyses this step. After formation of the peptide bond the tRNA carrying the peptide chain moves one codon along the mRNA, a process called 'tRNA translocation', conducted by elongation factor-G (EF-G). Then the elongation steps repeat and the cycle continues until a stop codon on mRNA reaches the decoding center. The stop codons signal release factors to bind on the mRNA and release the peptide from the tRNA. The process ends by splitting of the ribosomal subunits, which recycle for the next round of protein synthesis. The detailed mechanism of these steps although studied mainly with components from gram-negative bacteria *E. coli*, can be extrapolated to gram-positive mycobacteria due to high degree of sequence conservation in the translation components in all eubacteria. It should be mentioned that although the protein synthesis machinery is highly conserved in general, the fine differences that exist in this system between the pathogenic bacteria and the host organisms provide ample

opportunity for targeting the bacterial translation system in a rather specific manner.

Although in recent years, we have acquired a lot of knowledge on the mechanism of bacterial protein synthesis from high resolution crystal structure and fine-tuned biochemical

candidates for treatment of mycobacterial infections.

of not only tuberculosis, but also candidiasis and melioidosis and many other pathogens (Kumar, 2009). The rationing mechanism for supply of the precursor amino acid and nucleic acids required for growth can be halted and paused by inhibitors of metabolic pathways and thus making it tough for the bug to proliferate and persist. The search for elixir of life continues with the effort to evolve a drug that has bactericidal activity both on the active and persistent form and a robust delivery mechanism that has ability to penetrate diverse metabolic niche to eradicate these killer bugs.
