Isocitrate Succinate + Glyoxylate

Hence, this enzyme has one substrate, isocitrate, and two products, succinate and glyoxylate. This enzyme belongs to the family of lyases, specifically the oxo-acid-lyases, which cleave carbon-carbon bonds. The systematic name of this enzyme class is isocitrate glyoxylate-lyase (succinate-forming). Isocitrate lyase is the first enzyme unique to the metabolic pathway known as the glyoxylate cycle which is required for the assimilation of fatty acids and acetate (Kelly et al., 2002). Recent reports have alluded to an additional role for this enzyme in *M. tuberculosis* metabolism, specifically for growth on propionate. A product of betaoxidation of odd-chain fatty acids is propionyl-CoA. Clearance of propionyl-CoA and the

Acetyl-CoA + H2O + Glyoxylate (S)-Malate + CoA

Fig. 2. Reaction scheme of glyoxylate shunt.

*Mycobacterium tuberculosis*: Dormancy, Persistence and Survival in the Light of Protein Synthesis 223

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.

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

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

**3. The shunt and the hunt** 

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

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

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 (Sharma et al., 2000).
