The amino acid sequence of the motor domain of AnKinA, kinesin-1 from Aspergillus nidulans, growing optimally at 37°C, was compared with that of SbKin1, kinesin-1 from the snow mold Sclerotinia borealis. For cold-adaptation, some enzymes are thought to exhibit augmented protein structure flexibility, acquired most effectively by substituting a glycine residue for another amino acid residue. By the comparison described above, two glycine residues proximal to tightly bound ADP were identified in the SbKin1 motor domain, of which the corresponding residues of AnKinA were non-glycine ones (P60 and S323). We made AnKinA recombinant kinesin (AnKinA-WT (WT)) along with P60G and S323G mutants. From the basal ATPase activity (without microtubules), these kinesins showed similar characteristics in activation energies, while deviation from the linearity of the ATPase activity time-course was detected at 34°C for WT and P60G but at 24°C for S323G. The microtubule translocation velocity of WT, P60G or S323G exhibited an activation energy of 60, 58 or 53 kJ/mol, respectively; for S323G, the activation energy was lower and the velocity at low temperatures was higher than those for the other two. These results suggest that the point mutation S323G would offer possible cold-adaptation in compensation for thermal stability.
Keywords: activation energy; cold-adaptation; kinesin; temperature dependence; thermal stability.
© The Author(s) 2018. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.