Biochemical characterization of isocitrate dehydrogenase from Methylococcus capsulatus reveals a unique NAD+-dependent homotetrameric enzyme

Abstract

The gene encoding isocitrate dehydrogenase (IDH) of Methylococcus capsulatus (McIDH) was cloned and overexpressed in Escherichia coli. The purified enzyme was NAD+-dependent with a thermal optimum for activity at 55-60 degrees C and an apparent midpoint melting temperature (Tm) of 70 degrees C. Analytical ultracentrifugation (AUC) revealed a homotetrameric state, and McIDH thus represents the first homotetrameric NAD+-dependent IDH that has been characterized. Based on a structural alignment of McIDH and homotetrameric homoisocitrate dehydrogenase (HDH) from Thermus thermophilus (TtHDH), we identified the clasp-like domain of McIDH as a likely site for tetramerization. McIDH showed moreover, higher sequence identity (48%) to TtHDH than to previously characterized IDHs. Putative NAD+-IDHs with high sequence identity (48-57%) to McIDH were however identified in a variety of bacteria showing that NAD+-dependent IDHs are indeed widespread within the domain, Bacteria. Phylogenetic analysis including these new sequences revealed a close relationship with eukaryal allosterically regulated NAD+-IDH and the subfamily III of IDH was redefined to include bacterial NAD+- and NADP+-dependent IDHs. This apparent relationship suggests that the mitochondrial genes encoding NAD+-IDH are derived from the McIDH-like IDHs.

Nondecarboxylating and Decarboxylating Isocitrate Dehydrogenases: Oxalosuccinate Reductase as an Ancestral Form of Isocitrate Dehydrogenase

“…Prokaryotic ICDH in this group has previously been considered a homodimeric and an NAD(P)-dependent enzyme. However, due to increasing reports of NAD-dependent ICDH in bacteria (Acidithiobacillus, Aquifex, Hydrogenobacter, Methylophilus, and Streptococcus) and archaea (Pyrococcus) and of homotetrameric ICDH in bacteria (Methylococcus and Thermotoga) (3,6,7,9,(13)(14)(15)(22)(23), prokaryotic oligomeric ICDH is now recognized as an enzyme with various oligomeric states and coenzyme specificities.…”

“…Phylogenetic analyses of prokaryotic oligomeric ICDH indicate that this enzyme does not comprise a single lineage but can be divided into many subfamilies (21)(22)(23). EcICDH is one of the best analyzed forms and belongs to a distinctive subfamily that also contains ICDH from archaea (Aeropyrum, Archaeoglobus, Caldococcus, and Pyrococcus) and Aquificales (Aquifex and Hydrogenobacter) (3)(4)(5)(6)(21)(22)(23).…”

“…EcICDH is one of the best analyzed forms and belongs to a distinctive subfamily that also contains ICDH from archaea (Aeropyrum, Archaeoglobus, Caldococcus, and Pyrococcus) and Aquificales (Aquifex and Hydrogenobacter) (3)(4)(5)(6)(21)(22)(23). These enzymes can be considered a single lineage and can be categorized as EcICDH-type enzymes.…”

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

“…These homodimers may be described as pseudo 3D-domain-swapped dimmers [54,55] as a single subunit is not known to be independently active [4]. It has been speculated that higher order oligomers, such as tetramers [7,30] may exist, however they retain the homodimer as a basic unit. The prominent cross-over domain forming interaction between the two subunits is called the clasp domain as it resembles two hands, each representing a subunit, clasped together (see Figure 4 and Figure 5 for comparative structures).…”

“…We first extend earlier phylogenetic studies [6,8-10,30] using a larger number of sequences and combine this with structural information. Representative dimeric IDH structures were first aligned using the structural alignment tool STAMP [31] to ensure that functional residues (Table 1 for representative list) were aligned.…”