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.

Keywords: Acinetobacter baumannii; crystal structure; dimerization; isocitrate dehydrogenase; phylogenetic studies; thermostability.

Figure 1

Oligomeric state determination of the recombinant AbIDH2. The flow rate of the size exclusion chromatography was 0.5 mL·min⁻¹ and the proteins in the fractions were monitored at 280 nm. V_e of the recombinant AbIDH2 was 11.78 mL. The upper insert panel shows the protein purity detection by 12% SDS-PAGE. M, protein marker; lane 1, crude extracts of E. coli with pET-28b after isopropyl-β-d-thiogalactopyranoside (IPTG) induction; lane 2, crude extracts of E. coli with recombinant plasmid pET28-AbIDH2 after IPTG induction; lane 3, recombinant AbIDH2 after purification. The lower insert panel shows the sedimentation coefficient distribution of the recombinant AbIDH2 at 20 °C.

Figure 2

The overall structure of AbIDH2. (A) AbIDH2 shows a dimeric structure and each subunit contained a domain I and a domain II. (B,C) These images show the hydrophobic interactions formed on the interface platform that stabilize the dimeric assembly. The interface platform centers around the three parallel helices (α4, α12, and α17) and one adjacent loop from the domain II in each subunit.

Figure 3

Characterization of the mutant mAbIDH2. The flow rate of the size exclusion chromatography was 0.5 mL·min⁻¹ and the proteins in the fractions were monitored at 280 nm. V_e of the wild-type AbIDH2 and mutant mAbIDH2 were 11.78 and 14.35 mL, respectively. The upper insert panel shows the sequence alignment of the dimeric AbIDH2 with its homologous IDHs from Azoarcus tolulyticus (AtIDH, GenBank Accession WP_076602605.1) and Acidovorax ebreus (AeIDH, GenBank Accession WP_015913126.1) and two typical monomeric NADP-IDHs from Azotobacter vinelandii (AvIDH, GenBank Accession No. BAA11169.1) and Corynebacterium glutamicum (CgIDH, GenBank Accession No. WP_011013800.1). The residue numbering in the figure is based on the AbIDH2 sequence. The conserved residues are shaded in black. The residues that are involved in the dimer formation are marked by a pentagram ★. The lower insert panel shows the heat-inactivation profiles of the wild-type AbIDH2 and mutant mAbIDH2 from 25 to 47.5 °C.

Figure 4

Molecular phylogenetic analysis of the type III IDH protein subfamily by the Maximum Likelihood method. The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. The analysis involved 216 IDH sequences. All positions containing gaps and missing data were eliminated. Evolutionary analyses were conducted in MEGA7. The dimeric AbIDH2 and MtIDH2 (the second NADP-IDH from M. tuberculosis) were marked by closed circle (●) and triangle (▲), respectively.