Contribution of Three Different Regions of Isocitrate Dehydrogenases from Psychrophilic and Psychrotolerant Bacteria to Their Thermal Properties

Monomeric isocitrate dehydrogenases of a psychrophilic bacterium, Colwellia maris, and a psychrotolerant bacterium, Pseudomonas psychrophila, (CmIDH and PpIDH) are cold-adapted and mesophilic, respectively. On the other hand, previous studies revealed that the monomeric IDH of Azotobacter vinelandii (AvIDH) is also mesophilic and the regions 2 and 3 among three regions of this enzyme are involved in the thermal properties. Therefore, to examine whether the region(s) responsible for the mesophilic properties are common between PpIDH and AvIDH, the genes of chimeric IDHs exchanging three regions of PpIDH and CmIDH in various combinations were constructed and overexpressed as His-tagged recombinant proteins in the Escherichia coli cells, and the chimeric and wild-type PpIDH and CmIDH were purified with Ni-chelating affinity column chromatography. The swapping chimeras of the regions 2 or 3 in PpIDH and CmIDH showed lower and higher optimum temperatures for activities and their thermostabilities than the wild-type ones, respectively. On the other hand, the exchange of the respective region 1 hardly influenced these properties of the two IDHs. Therefore, the regions 2 and 3 of the two IDHs were confirmed to be involved in their thermal properties. These results were coincident with those of the previous study on chimeric IDHs between AvIDH and CmIDH, indicating that the common regions of AvIDH and PpIDH are responsible for their mesophilic properties and the amino acid residues involved in their thermal properties are present in the regions 2 and 3

ε Subunit of <i>Bacillus subtilis</i> F₁-ATPase Relieves MgADP Inhibition

<div><p>MgADP inhibition, which is considered as a part of the regulatory system of ATP synthase, is a well-known process common to all F₁-ATPases, a soluble component of ATP synthase. The entrapment of inhibitory MgADP at catalytic sites terminates catalysis. Regulation by the ε subunit is a common mechanism among F₁-ATPases from bacteria and plants. The relationship between these two forms of regulatory mechanisms is obscure because it is difficult to distinguish which is active at a particular moment. Here, using F₁-ATPase from <i>Bacillus subtilis</i> (BF₁), which is strongly affected by MgADP inhibition, we can distinguish MgADP inhibition from regulation by the ε subunit. The ε subunit did not inhibit but activated BF₁. We conclude that the ε subunit relieves BF₁ from MgADP inhibition.</p> </div

Effect of preincubation with MgADP.

<p>The α₃β₃γ or α₃β₃γε (5 µM) was incubated with the indicated concentrations of MgADP for more than 10 min at 25° C. Residual ATPase activity was measured in the presence of 2 mM ATP. The initial rate (2–4 s after the start of the reaction) was measured, and the values relative to the control without incubation with MgADP (82.9 ± 5.4 s^-1 and 88.6 ± 3.6 s^-1 for α₃β₃γ and α₃β₃γε, respectively) are plotted. Closed and open circles represent α₃β₃γ and α₃β₃γε, respectively. Error bars represent standard errors.</p

Kinetics of ATP hydrolysis by mutant α₃β₃γ^S3Cε^133C.

<p>ATPase activities of α₃β₃γ^S3Cε^133C (S3C), and α₃β₃γ^WTε^133C (WT) at 2 mM ATP were determined. The α₃β₃γε complex of was added to 3 nM at the times indicated by the first arrowheads. (<i>A</i>) DTT (50 mM) and LDAO (0.1%) were added at the times indicated by the second and third arrowheads, respectively. (<i>B</i>) The order of addition of DTT and LDAO was reversed.</p