Substrate‐free structure of a monomeric NADP isocitrate dehydrogenase: An open conformation phylogenetic relationship of isocitrate dehydrogenase

Abstract

Both monomeric and dimeric NADP+-dependent isocitrate dehydrogenase (IDH) belong to the metal-dependent beta-decarboxylating dehydrogenase family and catalyze the oxidative decarboxylation from 2R,3S-isocitrate to yield 2-oxoglutarate, CO2, and NADPH. It is important to solve the structures of IDHs from various species to correlate with its function and evolutionary significance. So far, only two crystal structures of substrate/cofactor-bound (isocitrate/NADP) NADP+-dependent monomeric IDH from Azotobacter vinelandii (AvIDH) have been solved. Herein, we report for the first time the substrate/cofactor-free structure of a monomeric NADP+-dependent IDH from Corynebacterium glutamicum (CgIDH) in the presence of Mg2+. The 1.75 A structure of CgIDH-Mg2+ showed a distinct open conformation in contrast to the closed conformation of AvIDH-isocitrate/NADP+ complexes. Fluorescence studies on CgIDH in the presence of isocitrate/or NADP+ suggest the presence of low energy barrier conformers. In CgIDH, the amino acid residues corresponding to the Escherichia coli IDH phosphorylation-loop are alpha-helical compared with the more flexible random-coil region in the E. coli protein where IDH activation is controlled by phosphorylation. This more structured region supports the idea that activation of CgIDH is not controlled by phosphorylation. Monomeric NADP+-specific IDHs have been identified from about 50 different bacterial species, such as proteobacteria, actinobacteria, and planctomycetes, whereas, dimeric NADP+-dependent IDHs are diversified in both prokaryotes and eukaryotes. We have constructed a phylogenetic tree based on amino acid sequences of all bacterial monomeric NADP+-dependent IDHs and also another one with specifically chosen species which either contains both monomeric and dimeric NADP+-dependent IDHs or have monomeric NADP+-dependent, as well as NAD+-dependent IDHs. This is done to examine evolutionary relationships.

A rheostat mechanism governs the bifurcation of carbon flux in mycobacteria

“…2d) similar to the short α-helix loop in CgICD and AvICD (Fig. 2b)2021. Overlaid with EcICD, this short α-helix limits access to the active site and would sterically hinder binding of AceK to the AceK recognition sequence (ARS) (Fig.…”

“…2a). A cleft region between the two domains contains the active site, as described for other ICD enzymes2021.…”

Structure of a highly NADP+-specific isocitrate dehydrogenase

“…Although the structure-based sequence homology between monomeric and dimeric IDHs is only 7-8%, the tertiary structure of monomeric IDHs (Yasutake et al, 2002;Imabayashi et al, 2006) is similar to that of the well studied dimeric EcIDH (Hurley et al, 1989) and the closely related isopropylmalate dehydrogenase (IPMDH; Imada et al, 1991). Residues binding the substrate isocitrate-Mn 2+ complex are also highly conserved between the two IDHs (Yasutake et al, 2002).…”

“…These include Azotobacter vinelandii (Chung & Franzen, 1969), Colwellia maris (Ochiai et al, 1979;Ishii et al, 1987), R. vannielii (Leyland & Kelly, 1991), Vibrio parahaemolyticus (Fukunaga et al, 1992) and Desulfobacter vibrioformis (Steen et al, 1998). Furthermore, genes or putative genes encoding monomeric IDHs have been described in approximately 50 bacterial species, including pathogenic bacteria such as Mycobacterium tuberculosis, M. leprae, Corynebacterium diphtheriae, Neisseria meningitidis, Vibrio cholerae, Francisella tularensis and Pseudomonas aeruginosa (Yasutake et al, 2002;Imabayashi et al, 2006). The glyoxylate-bypass enzyme isocitrate lyase (ICL) has been shown to help M. tuberculosis persist in mice (McKinney et al, 2000) and ICL and the two IDHs from the bacterium have been suggested as potential drug targets for tuberculosis (TB).…”

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

“…Subfamily I (S1-IDH) consists of homodimeric, prokaryotic and predominantly NADP-dependent IDHs. Subfamily II (S2-IDH)[9,53] consists of homodimeric, predominantly eukaryotic and NADP-dependent IDHs shown in Figure 4.…”