Comparison of Mycobacterium tuberculosis isocitrate dehydrogenases (ICD-1 and ICD-2) reveals differences in coenzyme affinity, oligomeric state, pH tolerance and phylogenetic affiliation

Abstract

BackgroundM.tb icd-1 and M.tb icd-2, have been identified in the Mycobacterium tuberculosis genome as probable isocitrate dehydrogenase (ICD) genes. Earlier we demonstrated that the two isoforms can elicit B cell response in TB patients and significantly differentiate TB infected population from healthy, BCG-vaccinated controls. Even though immunoassays suggest that these proteins are closely related in terms of antigenic determinants, we now show that M.tb icd-1 and M.tb icd-2 code for functional energy cycle enzymes and document the differences in their biochemical properties, oligomeric assembly and phylogenetic affiliation.ResultsFunctionally, both M.tb ICD-1 and ICD-2 proteins are dimers. Zn+2 can act as a cofactor for ICD-1 apart from Mg+2, but not for ICD-2. ICD-1 has higher affinity for metal substrate complex (Km (isocitrate) with Mg++:10 μM ± 5) than ICD-2 (Km (isocitrate) with Mg++:20 μM ± 1). ICD-1 is active across a wider pH range than ICD-2, retaining 33–35% activity in an acidic pH upto 5.5. Difference in thermal behaviour is also observed with ICD-2 being active across wider temperature range (20°C to 40°C) than ICD-1 (optimum temperature 40°C). The isozymes are NADP+ dependent with distinct phylogenetic affiliations; unlike M.tb ICD-2 that groups with bacterial ICDs, M.tb ICD-1 exhibits a closer lineage to eukaryotic NADP+ dependent ICDs.ConclusionThe data provide experimental evidence to show that the two open reading frames, Rv3339c (ICD-1) and Rv0066c (ICD-2), annotated as probable ICDs are functional TCA cycle enzymes with identical enzymatic function but different physio-chemical and kinetic properties. The differences in biochemical and kinetic properties suggest the possibility of differential expression of the two ICDs during different stages of growth, despite having identical metabolic function.

Structural, Kinetic and Chemical Mechanism of Isocitrate Dehydrogenase-1 from Mycobacterium tuberculosis

“…The importance of this interaction is highlighted by the inability of Mtb ICDH-1 to catalyze a detectable reaction using NAD + as its cofactor, as shown by Banerjee et al . (8) and repeated in our own lab (data not shown). These interactions are generally on the order of 3 Å, and we predict a collapse of the enzyme active site upon the binding of isocitrate in the cleft (Figure 5B) to strengthen these interactions.…”

“…Mtb has two ICDHs; the 45.5 kDa ICDH-1 and the 82.6 kDa ICDH-2, both of which are NADP + dependent (8) . Mtb ICDH-1 and ICDH-2 have differing kinetic parameters under several conditions including pH, temperature, and salt concentration, suggesting that each isoform may play a role in the diverse environmental conditions confronted by the bacilli (8) . However, no in vivo studies have been performed on the necessity of the two ICDHs, and the functional relevance of the two isoforms is still unknown.…”

A rheostat mechanism governs the bifurcation of carbon flux in mycobacteria

“…Characterization of this putative allosteric interaction and localization of the binding site of glyoxylate could provide new opportunities for therapeutic intervention, as flux partitioning between the TCA cycle and glyoxylate shunt is critical for survival of M. tuberculosis during infection. Similar to other NADP + -dependent ICDs, mycobacterial ICD2 forms dimers in solution1433. According to the ultrasensitivity theory34, such multimeric structures can mediate cooperative binding of substrates or allosteric effector molecules, thus enabling rapid and switch-like responses to environmental changes35.…”

“…The M. smegmatis genome encodes a single ICD (743 AA), a homologue of ICD2 in M. tuberculosis and M. bovis BCG. Both isoenzymes (ICD1 and ICD2) are biochemically active in vitro 14. To study the relative contributions of the isoenzymes to oxidative TCA cycle fluxes in vivo , we generated gene deletion strains.…”

Functional relevance of dynamic properties of Dimeric NADP-dependent Isocitrate Dehydrogenases

“…Examples of functional monomeric type are the Azotobacter vinelandii IDH [3] [PDB:1ITW] and Corynebacterium glutamicum IDH [PDB:2B0T]. Bacteria such as Mycobacterium tuberculosis [4] and Vibrio [5] have both dimeric type IDHs (IDH1) and monomeric type IDH (IDH2). Functionally dimeric IDHs are more abundant and diverse.…”