The “Catalytic” Triad of Isocitrate Dehydrogenase Kinase/Phosphatase from E. coli and Its Relationship with That Found in Eukaryotic Protein Kinases

Abstract

The isocitrate dehydrogenase kinase/phosphatase (IDHK/P) of E. coli is a bifunctional enzyme responsible for the reversible phosphorylation of isocitrate dehydrogenase (IDH) on a seryl residue. As such, it belongs to the serine/threonine protein kinase family. However, only a very limited homology with the well-characterized eukaryotic members of that family was identified so far in its primary structure. In this report, a new region of amino acids including three putative residues involved in the kinase activity of IDHK/P was identified by sequence comparison with eukaryotic protein kinases. In IDHK/P, these residues are Asp-371, Asn-377, and Asp-403. Their counterpart eukaryotic residues have been shown to be involved in either catalysis (former residue) or magnesium binding (the two latter residues). Site-directed mutagenesis was performed on these three IDHK/P residues, and also on the Glu-439 residue equivalent to that of the Ala-Pro-Glu motif found in the eukaryotic protein kinases. Mutations of Asp-371 into either Ala, Glu, or Gln residues drastically lowered the yield and the quality of the purification. Nevertheless, the recovered mutant enzymes were barely able to phosphorylate IDH either in vitro or after expression in an aceK (-) mutant strain. In contrast, mutation of either Asn-377, Asp-403, or Glu-439 into an Ala residue altered neither the yield of purification nor the maximal phosphorylating capacity of the enzyme. However, when IDH was phosphorylated in the presence of increasing concentrations of magnesium ions, the two former mutants displayed a much lower affinity for this cation, with a K(m) value of 0.6 or 0.8 mM, respectively, as compared to 0.1 mM for the wild-type enzyme. On the other hand, the Glu439Ala mutant has an affinity for magnesium essentially unaffected. Therefore, and in contrast to the current opinion, our results suggest that the catalytic mechanism of IDHK/P exhibits some similarities with that found in the eukaryotic members of the protein kinase family.

Sequence and Structure Classification of Kinases

“…These sequences can be identified by the presence of conserved active-site residues, which are well described for the protein kinase family, 8,9 and by the predicted secondary structure patterns ( Figure 1(b)). These proteins include hygromycin-B kinase, streptomycin 3 00 -kinase, fructosamine 3-kinase, isocitrate dehydrogenase kinase/ phosphatase, 10 kanamycin kinase, viomycin kinase, actin-fragmin kinase, homoserine kinase from several proteobacterial species, the kinase Figure 1. The PK family and the ATP-grasp family.…”

“…In addition to the ferredoxin-like core of NDP kinase, this enzyme has several other secondary structural elements, including the Kpn loop, an a-helix hairpin, and a C-terminal extension (Figure 4(a)). The Kpn loop is a small, compact structural element containing an interesting combination of helical structures: a turn of 3 10 helix, a turn of polyproline II lefthanded helix, and a turn of standard a-helix. 45 The Kpn loop and the a-helix hairpin constitute a nucleotide-binding site that is unique to NDP kinase (Figure 4(a)).…”

Proteomic investigation of the impact of oxygen on the protein profiles of hyaluronic acid‐producing Streptococcus zooepidemicus

“…It was implicated that the isoforms with lower pI might attribute to deamination or phosphorylation or to modification with a group resulting in a shift of the pI toward the acidic range of the 2-DE map [22]. Phosphorylation and dephosphorylation of proteins by kinases and phosphatases, respectively, has long been regarded as an essential mechanism by which the functional activities may be adjusted in eucaryotic cells but some of these modifications have now been observed in prokaryotic cells [23][24][25][26][27][28].…”

Structural and mechanistic insights into the bifunctional enzyme isocitrate dehydrogenase kinase/phosphatase AceK

“…Asp317, along with Asn377 and Asp403 completing the triad [11]. When the structure of AceK was solved, it was clear that this previously proposed 'catalytic triad' was incorrect.…”

“…The interface created between AceK and IDH is highly specific [11,48,49]. The SRL (residues 484 -510) of AceK deeply extends approximately 32 Å into the active site cleft of IDH with a short a-helix at its tip ( figure 7a).…”