homoisocitric-acid and isocitric-acid

β-Decarboxylating dehydrogenases, which are involved in central metabolism, are considered to have diverged from a common ancestor with broad substrate specificity. In a molecular phylogenetic analysis of 183 β-decarboxylating dehydrogenase homologs from 84 species, TK0280 from Thermococcus kodakarensis was selected as a candidate for an ancestral-type β-decarboxylating dehydrogenase. The biochemical characterization of recombinant TK0280 revealed that the enzyme exhibited dehydrogenase activities toward homoisocitrate, isocitrate, and 3-isopropylmalate, which correspond to key reactions involved in the lysine biosynthetic pathway, tricarboxylic acid cycle, and leucine biosynthetic pathway, respectively. In T. kodakarensis, the growth characteristics of the KUW1 host strain and a TK0280 deletion strain suggested that TK0280 is involved in lysine biosynthesis in this archaeon. On the other hand, gene complementation analyses using Thermus thermophilus as a host revealed that TK0280 functions as both an isocitrate dehydrogenase and homoisocitrate dehydrogenase in this organism, but not as a 3-isopropylmalate dehydrogenase, most probably reflecting its low catalytic efficiency toward 3-isopropylmalate. A crystallographic study on TK0280 binding each substrate indicated that Thr71 and Ser80 played important roles in the recognition of homoisocitrate and isocitrate while the hydrophobic region consisting of Ile82 and Leu83 was responsible for the recognition of 3-isopropylmalate. These analyses also suggested the importance of a water-mediated hydrogen bond network for the stabilization of the β3-α4 loop, including the Thr71 residue, with respect to the promiscuity of the substrate specificity of TK0280.

Topics: Archaeal Proteins; Catalytic Domain; Genetic Complementation Test; Isocitrates; Lysine; Malates; Oxidoreductases; Protein Structure, Secondary; Recombinant Proteins; Structure-Activity Relationship; Substrate Specificity; Thermococcus; Thermus thermophilus; Tricarboxylic Acids

The kinetic mechanism of homoisocitrate dehydrogenase from Saccharomyces cerevisiae was determined using initial velocity studies in the absence and presence of product and dead end inhibitors in both reaction directions. Data suggest a steady state random kinetic mechanism. The dissociation constant of the Mg-homoisocitrate complex (MgHIc) was estimated to be 11 +/- 2 mM as measured using Mg2+ as a shift reagent. Initial velocity data indicate the MgHIc complex is the reactant in the direction of oxidative decarboxylation, while in the reverse reaction direction, the enzyme likely binds uncomplexed Mg2+ and alpha-ketoadipate. Curvature is observed in the double-reciprocal plots for product inhibition by NADH and the dead-end inhibition by 3-acetylpyridine adenine dinucleotide phosphate when MgHIc is the varied substrate. At low concentrations of MgHIc, the inhibition by both nucleotides is competitive, but as the MgHIc concentration increases, the inhibition changes to uncompetitive, consistent with a steady state random mechanism with preferred binding of MgHIc before NAD. Release of product is preferred and ordered with respect to CO2, alpha-ketoadipate, and NADH. Isocitrate is a slow substrate with a rate (V/E(t)) 216-fold slower than that measured with HIc. In contrast to HIc, the uncomplexed form of isocitrate and Mg2+ bind to the enzyme. The kinetic mechanism in the direction of oxidative decarboxylation of isocitrate, on the basis of initial velocity studies in the absence and presence of dead-end inhibitors, suggests random addition of NAD and isocitrate with Mg2+ binding before isocitrate in rapid equilibrium, and the mechanism approximates rapid equilibrium random. The Keq for the overall reaction measured directly using the change in NADH as a probe is 0.45 M.

Topics: Alcohol Oxidoreductases; Isocitrates; Kinetics; Magnesium; Saccharomyces cerevisiae; Tricarboxylic Acids