Comparison of Mycobacterium tuberculosis isocitrate dehydrogenases (ICD-1 and ICD-2) reveals differences in coenzyme affinity, oligomeric state, pH tolerance and phylogenetic affiliation
© Banerjee et al; licensee BioMed Central Ltd. 2005
Received: 08 February 2005
Accepted: 29 September 2005
Published: 29 September 2005
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.
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.
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.
The central metabolic pathways in bacteria, especially in E.coli, have been extensively studied to understand the physiology of the organisms under altered carbon sources . One of the key regulatory enzymes in the universal tri-carboxylic acid energy cycle is the isocitrate dehydrodenase that allosterically regulates the conversion of oxidative decarboxylation of D-isocitrate to α-ketoglutarate and CO2 in presence of a cofactor . This rate-limiting step is the first NADPH yielding reaction of the TCA cycle . Isocitrate dehydrogenase belongs to a family of enzymes that exhibits diversity with regard to amino acid composition, cofactor specificity, metal ion requirement and oligomeric state. NADP-linked ICDs have been purified and studied from a variety of eukaryotes and prokaryotes with detail investigations on their subunit composition and kinetic properties [3–11]. ICD from different organisms has been phylogenetically affiliated to three subfamilies . Majority of the bacterial ICDs fall into subfamily I that includes archaeal and bacterial NADP dependent ICDs.
M. tb genome has two isoforms of isocitrate dehyrogenase, Rv3339c (ICD-1) and Rv0066c (ICD-2), both annotated as probable isocitrate dehydrogenase based on homology with other enzymes of the ICD family . The two isoenzymes are share only ~14% identity at amino acid level. Earlier, we have pointed to a very unusual property of this TCA cycle enzyme demonstrating that the two isoforms can elicit B cell response in TB patients and significantly discriminate healthy, BCG-vaccinated controls from different groups of TB-infected population when compared to PPD and control antigen M.tb HSP 60 . Although the two isoforms have primarily similar antigenic properties, little is known about their enzymatic properties. We now document differences in their biochemical properties, subunit composition and phylogenetic association. Our study provides experimental evidence to show that the two ORFs are TCA cycle enzymes with identical enzymatic function but different physio-chemical and kinetic properties.
Expression, purification and quantification of M.tb ICD-1 and ICD-2
The ORFs encoding hypothetical protein M. tb ICD-1 (Rv3339c) and the probable ICD2 (Rv0066c) are functionally expressed as evident from serological evidences
The M. tb icd-1 and M. tb icd-2, both are annotated as the probable isocitrate dehydrogenase based on the sequence homology with ICD-family of enzymes. Serological evidences reveal the presence of antibody titers against both the purified proteins ICD-1 and ICD-2 in the infected sera samples . Figure 1 is a sample representation of ten patients each for ICD-1 (sample 1–10) and ICD-2 (sample 11–20) with their respective healthy controls (Figure 1; 21–25, control reactions for ICD-1 and 26–30, control reactions for ICD-2) suggesting that both the ORFs encoding ICDs are expressed at the protein level as evident from antibodies in TB infected patient sera.
Biochemical characterization reveals differences between ICD-1 and ICD-2 in terms of pH and heat stability
In order to determine the kinetic parameters, the stabilizing components in the reaction like pH, temperature, salt requirement, metal ion components and coenzyme specificity were tested for each enzyme for optimal activity.
The decrease in the activity at higher salt concentration indicates involvement of ionic interactions during catalysis of M. tb ICD-1 and ICD-2
M. tb ICD-1 and M. tb ICD-2 are NADP-dependent and have differential metal cofactors requirement
Kinetic parameters and feedback inhibition of M. tb ICDs
Kinetic parameters for M. tb ICD-1 and M. tb ICD-2 with respect to Mg++ and Zn++
M. tb ICD-1
10 μM ± 5
22 μM ± 7
380 μM NADPH/min
190 μM NADPH/min
3.8 μM NADPH/min/pM enzyme
1.9 μM NADPH/min/pM enzyme
125 μM ± 5
400 μM NADPH/min
4 μM NADPH/min/pM enzyme
M. tb ICD-2
20 μM ± 1
371.3 μM NADPH/min
37.13 μM NADPH/min/pM enzyme
19.6 μM ± 6
374 μM NADPH/min
37.4 μM NADPH/min/pM enzyme
0.46 × 10-5 M.
Oligomeric assemblies of M. tb ICDs
We also performed chemical crosslinking in presence and absence of coenzyme (NADP) and substrate (Isocitrate). A band corresponding to a trimer was consistently present with the dimeric band (figure 10c). Since the gel filtration data under low salt condition showed a tendency of forming lower oligomeric states, we further explored if under any salt condition a trimeric species that was observed in crosslinking could be obtained. Therefore the gel filtration assay was repeated under an intermediate (500 mM) salt concentration. Figure 10d shows a peak representing a mixture of trimeric and dimeric species under this buffer condition. Therefore, we conclude that M. tb ICD-2 is not a monomeric protein under any conditions tested in this study. Eventhough it can exist in higher oligomerc forms, dimer represented the most stable form that could not be further dissociated into monomers in our buffer condition. The above result equivocally demonstrate that M. tb ICD-2 is not a monomer and exists in higher oligomeric state in contrary to what has been reported in the annotation of H37Rv genome.
M. tb ICD-1 and M. tb ICD-2 have different phylogenetic affiliations
Our results demonstrate for the first time experimentally that the two M. tb ORFs Rv3339c and Rv0066c code for functional TCA cycle enzyme that can catalyze the conversion of D-isocitrate to α-ketoglutarate and CO2 in presence of NADP as cofactor and differ in their biochemical properties. Interestingly, an altogether different functionality of these two isoforms in immune recognition was evident from our earlier work . We have biochemically characterized the two isoforms of M. tb ICDs and established the differences between them.
Isocitrate dehydrogenase is an important regulatory enzyme of TCA cycle that has been intensively studied in both prokaryotes and eukaryotes (3–11). It lies at the branchpoint between glyoxylate shunt and citric acid cycle in prokaryotes where the switchover from TCA cycle to glyoxylate shunt depends upon the alteration in the biochemical parameters of isocitrate dehydrogenase. Isocitrate lyase of glyoxylate shunt pathway has much lower affinity for the isocitrate and cannot compete with isocitrate dehydrogenase for the substrate under normal conditions. It has been reported that phosphorylation of ICD controls the flux of isocitrate between the Krebs cycle and the glyoxylate pathway [15, 16]. In E.coli, where glyoxylate bypass and citric acid cycle operate concurrently, the activity of a single, functional isocitrate dehydrogenase is closely monitored [17, 18]. In M. tb, however, glyoxylate bypass is observed inside macrophages where C2 substrate is the main carbon source . The occurrence of two isoforms of ICD in M. tb genome with the possibility of each having characteristic biochemical properties is interesting under such circumstances.
Comparison of NADP+-dependent isocitrate dehydrogenases from different organisms.
45 μM ± 13
27 μM ± 10
5.9 μM ± 0.9
14.5 μM ± 2.2
4.9 μM ± 0.2
19.6 μM ± 3.6
Synechocystis sp PCc 6803
Mycobacterium phlei (ATCC-354)
M. tb ICD-1
10 μM ± 5
125 μM ± 5
M. tb ICD-2
20 μM ± 1
19.6 μM ± 6
Beef liver NADP+-IDH
Rat liver (cytosolic)
9.7 μM ± 2.9
11.5 μM ± 0.2
Porcine heart NADP+ IDH
5 μM ± 0.19
The fact that M. tb ICD-1 could tolerate a broad range of both pH and temperature (Figure 2, 3) than M. tb ICD-2 indicates its robustness. The difference in the pH tolerance helps to postulate the possibility of differential expression of the two isoforms with ICD-1 being expressed during stationary phase when the intracellular pH is expected to vary over a wider range than log phase.
Km (NADP+) of M. tb ICD-1 showed poor affinity for NADP+ as compared to M. tb ICD-2 and other known NADP-dependent isocitrate dehydrogenases (Table 2). The poor affinity of M. tb ICD-1 to NADP+ warranted an investigation on whether dual co-enzyme specificity occurs in M. tb ICD-1 as reported in some archaeal bacteria . We, therefore, compared the enzymatic activity of both the enzymes in presence of NADP+ and NAD+ (Figure 5a and 5b). It can be clearly seen that M. tb ICD-1 as well as M. tb ICD-2 accepts NADP+ and not NAD+ as a proton acceptor.
The homodimeric state of M. tb ICD-1 is the functionally active species, even though residual activity was noticed in tetrameric fraction which could be a reflection of the presence of a few dimeric species as a consequence of disintegration of the tetrameric forms. The chromatogram peak for M. tb ICD-2 corresponding to a tetramer (~320 KDa) under low salt condition, which was dissociated into a dimer (~180 KDa) at a high salt concentration of 1 M, but not into a monomer (Figure 9) provided a strong evidence that the most stable form of M. tb ICD-2 is a dimer. An intermediary trimeric form was observed in chemical crosslinking assays (Figure 10b and 10c), both in presence and absence of coenzyme NADP and substrate isocitric acid. The data were consistent with the gel filtration profile under an intermediate (500 mM) salt concentration (Figure 10d) where it showed a peak representing a mixture of trimeric and dimeric species. We therefore could conclude that M. tb ICD-2 is not a monomeric protein. Our result indicates M. tb ICD-2 exists in different higher oligomeric states which may follow the following equilibria: [tetramer] ⇔ [trimer] ⇔ [dimer]. However, the physiological relevance of the different oligomers could not be concluded from our experiments.
Earlier attempt to trace the evolution of ICDs to understand the adaptive role of isocitrate dehydrogenase in intracellular persistence of this pathogen by Steen et al  does not place M. tb ICD-2 phylogenetically. Proximity of the two M. tb ICDs with other isocitrate dehydrogenases was determined. Our results on phylogenetic analysis of M. tb ICD-1 revealed a closer relationship with eukaryotic NADP+ dependent ICDs (Figure 11) with more than 65% identity with that of Glycine max, Sus scrofa, Bos and Homo sapiens. M. tb ICD-1, indeed, is correctly placed in subfamily II that includes eukaryotic NADP dependent ICDs and a single bacterial ICD (Sphingomonas yanoikuyae) . With NADP+ dependent isocitrate dehydrogenase of Sphingomonas yanoikuyae, M. tb ICD-1 has more than 65% similarity at primary structure level. Phylogenetic analysis of M. tb ICD-2 showed that the classical nomenclature applies to ICD-2 and it can be placed in subfamily I, the closest being M. leprae (Figure 12). The closest bacterial relative of M. tb ICD-1 as inferred by our study is NADP dependent isocitrate dehydrogenase of Bifidobacterium longum (Figure 11). Bifidobacterium sp. are gram positive, anaerobic, natural components of human intestinal microbiota . This might be argued as a case of horizontal transfer or lateral transfer of gene amongst unrelated organisms across the boundaries of phylogenetic domains. Horizontal transfer of genes is a common occurrence in nature and accounts for almost 10–50% of genes in bacteria [21, 22].
Several ORFs have been characterized since deciphering of M. tb genome [23–25]. Our data represent conclusive proof that the two ORFs, Rv3339c and Rv0066c, are functional TCA cycle enzyme and represent the first attempt to characterize these important members of the TCA cycle of Mycobacterium tuberculosis. Our studies conclusively reveal that both ICD-1 and ICD-2 are NADP+ dependent members of ICD family with the former having closer homology with eukaryotic ICDs and latter with prokaryotes. ICD-1 is a homodimer, while ICD-2 annotated as a monomer, exists in higher oligomeric forms, the dimer being the most stable. The two isoforms differ in their affinity for coenzyme NADP as represented by their Km(NADP) values (Table 1) and also with respect to pH tolerance and thermostability.M. tb ICD-2 is a more efficient enzyme as inferred by comparing Vmax(NADP)/Km(NADP) ratios for the two enzymes but M. tb ICD-1 is more robust in terms of pH tolerance and thermostability. The possibilities of differential expression of these two isoforms during different stages and conditions of growth cannot be ruled out even though the two isoforms have identical enzymatic function.
Cloning and purification of M. tb ICDs
The 1.230 kb (ICD-1) and 2.238 kb (ICD-2) long ORF was amplified from H37Rv genomic DNA and overexpressed in the pRSET-A/E.coli BL-21 (DE3) expression system as described earlier . The overexpressed his-tagged recombinant protein was purified by Ni2+-nitrilotriacetate affinity chromatography.
Enzyme linked immunosorbent assays (ELISA)
ELISAs were performed with purified recombinant proteins, as described earlier , to check the B cell immune response in TB patient sera as evidence to the in vivo expression of the proteins.
Dehydrogenase kinetics/biochemical assays
Dehydrogenase activity was measured spectrophotometrically by monitoring the time dependent reduction of NADP+ to NADPH at 25°C in Unicam UV/Vis spectrometer at 340 nm, the absorbance maximum of NADPH. The standard assay solution contained 20 mM triethanolamine chloride buffer pH 7.5, 2 mM NADP+, 0.03 mM DL-isocitrate, 10 mM MgCl2/10 mM ZnCl2, 100 mM NaCl and 10 -100 pM of the enzyme in a final volume of 400 μL. Environmental parameters for the enzymes were measured by altering the pH of the buffer (range 4 – 10), temperature (20 – 65°C), concentration of substrate (0.01 mM – 0.18 mM), cofactor (0.1 – 2 mM), metal ion (Mg++, Zn++, Mn++; 1 – 12.5 mM) and salt (100 mM – 500 mM) requirement (as indicated in the respective figure legends. The pH dependence of the enzyme was measured using the following buffers: 30 mM Na-acetate buffer (pH 4.0 to pH 5.5), 20 mM phosphate buffer (pH 5.7 to pH 7), 30 mM imidazole buffer (pH 6 to pH 7) and 20 mM Tris buffer (pH 7.5 to pH 10). The cofactor specificity was checked with both NADP+ and NAD+. The heat denaturation was studied in 20 mm triethanolamine chloride buffer pH 7.5 in presence of 1% bovine serum albumin. Enzyme aliquotes were placed in tubes and incubated for 30 minutes in a water bath set at the required temperature (20°C – 65°C). After heating, aliquotes were immediately placed on ice and then assayed for remaining enzyme activity.
The kinetic analysis was carried out at 25°C and pH 7.5 in presence of either Mg++ or Zn++. Km was determined by altering the concentration of either the substrate or the coenzyme. The substrate concentration gradient varied from 0.01 mM to 0.75 mM, while NADP+ concentration was taken from 0.1 mM to 2 mM. The values were plotted as V vs S for calculating Km and Vmax for this first order reaction. The results were counter checked by double inverse Lineweaver-Burk plot. Competitive inhibition was observed with reduced NADP (NADPH) versus NADP to estimate inhibitor constant, Ki. Standard Km analysis was performed followed by repeating the assay with NADPH. Two concentrations of the inhibitor were tested, 0.002 mM and 0.005 mM. The uninhibited run provided the value of Km for the reaction and the inhibited run provided the apparent Km (Kmapp) for the reaction. Ki for the competitive inhibition was calculated by the formula (Km) (I)/Kmapp – Km.
Size exclusion chromatography
Size exclusion chromatography was performed at room temperature using FPLC equipped with Superdex-200 HR 10/30 column (Amersham Pharmacia Biotech) and SuperoseTM 6 10/300 GL (BioRad BioLogic Duo-flow™). Calibration of the columns were performed using protein molecular-mass standards for gel-filtration (Sigma, USA) as described elsewhere . The void volume (Vo) was determined by running Blue Dextran on the column. The calibration curve was plotted as Ve/Vo versus log of molecular mass. A 2.4 mg/ml (for ICD-1) and 1.06 mg/ml (for ICD-2) concentrations of recombinant proteins were used for all gel filtration experiments. The columns were equilibrated with three bed volumes of the elution buffer prior to each run. Protein elution was monitored at A280.
UV and chemical crosslinking
UV crosslinking assays were performed to check the oligomeric assembly of M. tb ICD2. 5 μg of protein per reaction, in TrisCl buffer pH 8, was taken and exposed to UV in a UVP CL-1000 Ultraviolet crosslinker for 1 to 10 minutes at the rate of 1600 Joules/minute and fractioned later on 10% SDS-PAGE along with similar amount of untreated M. tb ICD2 as control. For chemical crosslinking, the protein was equilibrated in 20 mM phosphate buffer pH7.8. 10 mM Glutaraldehyde was used for all the chemical crosslinking reactions. The protein samples were incubated at 37°C, with or without glutaraldehyde. The reaction was stopped at different time points (10' or 20') using SDS loading dye containing 400 mM glycine and subsequently fractionated on 7% SDS-PAGE. 1 mM of either NADP or Isocitrate was used wherever required.
Sequence alignment and phylogenetic analysis
The amino acid sequence of M.tb ICD-1 and M.tb ICD-2 were compared against the NCBI protein database . The sequences with the BLAST score upto e-153 or 65% identity were selected for construction of the phylogenetic tree. The sequences were aligned using CLUSTAL program. Manual alignment was done by Jalview  wherever required. The sequence alignment is available on request from the authors. Rooted phylogenetic tree were constructed using the software MEGA3  using the amino acid sequence of Thermotoga maritima isocitrate dehydrogenase as outgroup. The confidence was assessed by bootstrap analysis (thousand replicates using default parameters).
List of abbreviations
nicotinamide adenine dinucleotide phosphate
Bovine Serum Albumin
nicotinamide adenine dinucleotide
reduced nicotinamide adenine dinucleotide phosphate
kilo Daltons, pM, pico molar
This work was supported by research grants to SEH from the Council of Scientific and Industrial Research (CSIR) and Department of Biotechnology, Government of India. S B was supported by a Senior Research Fellowship from CSIR. We thank Dr. Ranjan Sen and Dr. Shekhar C Mande, Centre for DNA Fingerprinting and Diagnostics for helpful discussions. We wish to thank Sriramana, Laboratory of Molecular Genetics, Bibhusita, Jisha, Nancy and Irfan of Transcriptional Biology Laboratory, CDFD for their help and co-operation.
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