Glycerol kinases from Thermococcus kodakaraensis KOD1 (Tk-GK) and Escherichia coli (Ec-GK) greatly differ in thermostability. The temperature (T-1/2) at which the enzymes lose half of their activity upon incubation for 20 min is 50-55 degreesC for Ec-GK and similar to 95 degreesC for Tk-GK. To examine whether the amino acid substitutions that make Tk-GK more stable than Ec-GK are localized in a limited region, the chimeras of two parental genes encoding Tk-GK and Ec-GK were constructed by DNA shuffling. E. coli cells were transformed with a plasmid library harboring these chimeras and screened for those that produce chimeric enzymes which are more stable than Ec-GK. Four chimeric enzymes were isolated and purified, and their biochemical properties characterized. Replacement of 83 or 93 residues in the C-terminus of Ec-GK with the corresponding ones of Tk-GK increased the T-1/2 value of Ec-GK by 25-30 degreesC. In contrast, replacement of 85 residues in the N-terminus of Ec-GK with the corresponding ones of Tk-GK reduced the T-1/2 value by 5-10 degreesC. In addition, replacement of 10 residues in the C-terminus of Tk-GK with the corresponding ones of Ec-GK reduced the T-1/2 value of Tk-GK by similar to 15 degreesC. Measurement of the far-UV CD spectra indicates that the three-dimensional structures of the chimeric enzymes, as well as those of the parent enzymes, are similar to one another. These results suggest that the amino acid substitutions responsible for the high stability of Tk-GK are largely localized in the C-terminal region.