Crystal Structure of the Isocitrate Dehydrogenase 2 from Acinetobacter baumannii (AbIDH2) Reveals a Novel Dimeric Structure with Two Monomeric-IDH-Like Subunits

Abstract

Monomeric isocitrate dehydrogenases (IDHs) have a single polypeptide sizing around 85 kDa. The IDH2 from the opportunistic bacterium Acinetobacter baumannii (AbIDH2) with a mass of 83 kDa was formerly recognized as a typical monomeric IDH. However, both size exclusion chromatography and analytical ultracentrifugation analysis indicated that AbIDH2 exists as a homodimer in solution. The crystallographic study of the substrate/coenzyme-free AbIDH2 gave a dimeric structure and each subunit contained a domain I and a domain II. The dimeric assembly is mainly stabilized by hydrophobic interactions (16 hydrogen bonds and 11 salt bridges) from the dimer’s interface platform, which centered around the three parallel helices (α4, α12, and α17) and one loop from the domain II. Kinetic analysis showed that the dimeric AbIDH2 showed much lower catalytic efficiency (0.39 μM−1·s−1) as compared to the typical monomeric IDHs (~15 μM−1·s−1). Key residues crucial for dimer formation were simultaneously changed to generate the mutant mAbIDH2. The disruption of the hydrophobic forces disassociated the dimeric AbIDH2, making mAbIDH2 a monomeric enzyme. mAbIDH2 sustained specific activity (21.9 ± 2 U/mg) comparable to AbIDH2 (25.4 ± 0.7 U/mg). However, mAbIDH2 proved to be a thermolabile enzyme, indicating that the thermostable dimeric AbIDH2 may have a physiological significance for the growth and pathogenesis of A. baumannii. Phylogenetic analysis demonstrated the existence of numerous AbIDH2 homologous proteins, thus expanding the monomeric IDH protein family.

Biochemical and Phylogenetic Characterization of a Novel NADP+-Specific Isocitrate Dehydrogenase From the Marine Microalga Phaeodactylum tricornutum

“…Additionally, the K m values of PtIDH2 for NADP + were 37.37 ± 4.02 μM with Mn 2+ and 18.37 ± 2.94 μM with Mg 2+ , and the corresponding catalytic efficiency ( k cat / K m ) values were 3.22 and 2.36 μM −1 s −1 ( Supplementary Figure S2 ). These k cat / K m values were similar to those for the bacterial monomeric NADP-IDHs from Xanthomonas campestris (6.0 μM −1 s −1 with Mn 2+ ) and Streptomyces avermitilis (11.7 μM −1 s −1 with Mn 2+ ) ( Wang et al, 2011 ; Lv et al, 2016 ), but higher than those for the homodimeric NADP-IDH (contains two monomeric IDH-like subunits) from A. baumannii (0.39 μM −1 s −1 with Mn 2+ ) ( Wang et al, 2018 ) and lower than those for the monomeric IDHs from A. vinelandii (15.0 μM −1 s −1 with Mn 2+ ) and Xylella fastidiosa (96.5 μM −1 s −1 with Mn 2+ ) ( Watanabe et al, 2005 ; Lv et al, 2018 ).…”

“…Based on phylogeny, the IDH protein family has been classified into three subfamilies, namely, types I, II, and III. Additionally, two types of IDHs—monomeric and homodimeric NADP-IDHs—can be distinguished in the type III subfamily based on coenzyme specificity and oligomeric state ( Wang et al, 2015 ; Wang et al, 2018 ). All type III IDHs reported to date are derived from prokaryotes, including both bacteria and archaea ( Wang et al, 2020 ).…”

“…Type I IDHs comprise bacterial and archaeal homodimeric NAD(P)-IDHs, bacterial homotetrameric NAD-IDHs, and eukaryotic mitochondrial heterooligomeric NAD-IDHs; type II IDHs are mainly comprised of bacterial homodimeric NADP-IDHs, bacterial homohexameric NAD-IDHs, and eukaryotic mitochondrial and cytoplasmic homodimeric NADP-IDHs; meanwhile, type III IDHs encompass all prokaryotic monomeric NAD(P)-IDHs. Notably, we recently reported a group of homodimeric NADP-IDHs from pathogenic bacteria, such as Acinetobacter baumannii , that contain two monomeric IDH-like subunits and belong to the type III IDH subfamily ( Wang et al, 2018 ). Although type I and II IDHs display similar subunit molecular mass (40–45 kDa) and three-dimensional structures, their amino acid sequence identities are very low (<15%).…”

Biochemical Characterization and Crystal Structure of a Novel NAD+-Dependent Isocitrate Dehydrogenase from Phaeodactylum tricornutum

“…IDHs are widely distributed in living organisms, including bacteria, archaea, and eukaryotes. A phylogenetic analysis revealed that the IDH protein family can be divided into three subfamilies: type I, type II, and type III [ 10 , 11 ]. All archaeal and the vast majority of bacterial homodimeric NAD(P)-IDHs and eukaryotic hetero oligomeric NAD-IDHs are grouped together in the type I subfamily.…”

“…In contrast to the type I and II IDHs with 400-amino acid polypeptide chains, the bacterial monomeric NAD(P)-IDHs belonged to the type III subfamily, the polypeptide chains of which are approximate 740-amino acid in length. We demonstrated in a previous study that some monomeric IDHs exist as dimers in solution, thus expanding the diversity of the IDH family [ 10 ].…”