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

Abstract

Background: M.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. Results: Functionally, 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. Conclusion: The 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.