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

Abstract

Background: Isocitrate Dehydrogenases (IDHs) are important enzymes present in all living cells. Three subfamilies of functionally dimeric IDHs (subfamilies I, II, III) are known. Subfamily I are well-studied bacterial IDHs, like that of Escherischia coli. Subfamily II has predominantly eukaryotic members, but it also has several bacterial members, many being pathogens or endosymbionts. subfamily III IDHs are NAD-dependent. The eukaryotic-like subfamily II IDH from pathogenic bacteria such as Mycobacterium tuberculosis IDH1 are expected to have regulation similar to that of bacteria which use the glyoxylate bypass to survive starvation. Yet they are structurally different from IDHs of subfamily I, such as the E. coli IDH.

Results: We have used phylogeny, structural comparisons and molecular dynamics simulations to highlight the similarity and differences between NADP-dependent dimeric IDHs with an emphasis on regulation. Our phylogenetic study indicates that an additional subfamily (IV) may also be present. Variation in sequence and structure in an aligned region may indicate functional importance concerning regulation in bacterial subfamily I IDHs. Correlation in movement of prominent loops seen from molecular dynamics may explain the adaptability and diversity of the predominantly eukaryotic subfamily II IDHs.

Conclusion: This study discusses possible regulatory mechanisms operating in various IDHs and implications for regulation of eukaryotic-like bacterial IDHs such as that of M. tuberculosis, which may provide avenues for intervention in disease.

Figure 1

Phylogenetic tree from UPGMA method. Phylogenetic tree calculated using UPGMA Method. The tree diagram shows phenetic relationship. The alignment used is provided by Additional file 1. The reference table is in Additional file 2.

Figure 2

Phylogenetic tree from Maximum likelihood. Phylogenetic tree calculated using Maximum likelihood Method. The tree diagram shows phylogenetic relationships. The alignment used is provided by Additional file 1. The reference table is in Additional file 2.

Figure 3

Alignment of dimeric IDH sequences. This is an alignment of sequences given in Table 1. Numbers correspond to residues given in Table 2. The numbers are 1-9 and A-F. Colors correspond to those given in structure markers in other figures. Some C-terminal residues of Thermus thermophilus TtIDH are not shown, as this IDH is longer than other IDHs and the extra region doesn't align with the other IDH sequences.

Figure 4

Structures of subfamily I and II. Structures of subfamily I (top) and II (bottom)are shown for comparison. Colors are consistent with regions in Figure 3. Note the difference in Clasp region, the three loops and the ARS-like region. Subfamily I IDHs have α-helices (β-α-β pattern from each subunit). Subfamily II have all β (β-ββ-β) greek-key motif [57,58]. Images were made using Chimera [80].

Figure 5

Structures of subfamily III and IV. Structures of subfamily III (top) and IV (bottom)are shown for comparison. Colors are consistent with regions in Figure 3. The sequentially central homologous clasp region (C1) in subfamilies III and IV is reduced to a two-strand anti-parallel sheet (ββ) (residues 148-160 in TtIDH), and is similar in both. C-terminal forms a larger domain over the clasp (C2). Images were made with Chimera [80].

Figure 6

ARS-like segments in various IDHs. The AceK recognition segment (ARS) in E.coli IDH [22] and ARS-like region sequences and structures in other IDHs. S1-IDHs have at least five groups with different structures, three of which are structurally represented here. Cyanobacteria like Nostoc IDH_ANASP have the longest ARS-like sequence, which is not structurally resolved yet. The shortest S1-type, IDH_STRMU (Streptococcus mutans) may be NAD-dependent. S2-IDHs have conserved structure, represented by Pig PmIDH. The residues may differ, however, as the alignment between PmIDH and Mycobacterium tuberculosis IDH_MYCTU shows here. The MtIDH sequence has a stretch of glutamates (-EEE-) and is richer in acidic residues. The shortest length is seen TtIDH, as well as S3-IDHs. Image was made using Chimera [80] and Jalview [33].

Figure 7

Fluctuations of IDHs. Fluctuations of dimeric IDH. (a) E. coli (EcIDH) and (b) Sus scrofa (PmIDH). The colored regions correspond to alignment in Figure 3 and regions in 4. Note that loops in PmIDH have helix structures within them. The numbering is continuous for the whole dimeric protein - subunit boundary is marked by thin black line in centre.

Figure 8

Correlation map for S1-IDH. Normalized Correlation map representative for dimeric S1-IDH (E.coli). The symmetric correlation matrix has been split, with lower triangle showing only negative values and upper triangle showing only positive values. Numbering of residues is continuous for each dimer (1- > ~800).

Figure 9

Correlation map for S2-IDH. Normalized Correlation map representative for dimeric S2-IDH (Sus scrofa mitochondrial). S2-IDH map has been annotated. Colored circles within the lower triangle region representing negative correlations, show the general movement indicated in the inset image, with the color bars corresponding to the color codes in Figure 3, Figure 4 and Figure 7. The region highlighted in the upper triangle of the matrix show the positive correlations of the loops with each other (green) and the central region (blue). This graph was plotted using Bio3d [52] and structure image was made in Chimera [80].

Figure 10

Principal Component analysis. Porcupine plots [59] for (a) EcIDH and (b)PmIDH. Only Cα atoms are shown for First PCA mode. The loop present at top and bottom of structure is the ARS region. Subfamily I show localized loop motion in a rotatory fashion around the central domain. Subfamily II shows tandem motion - as one site closes, the other opens. The loops are mobile, and may play a role to guide substrate and cofactor to the active site. The summary plots are provided [see Additional file 4].

Figure 11

Correlation map for MtIDH1. The region around S213, including the ARS-like region just above it, shows negative correlations not seen in any S2-type IDH simulated here. The ARS-like region in particular shows negative correlations, and so does S213 and its immediate vicinity. This movement may be biologically relevant, as it does not appear in any other IDH simulation, particularly S2-IDHs, and is unlikely to be obtained by chance.