Effects of C-terminal deletion on the activity and thermostability of orotate phosphoribosyltransferase from Thermus thermophilus.
To investigate the role of the C-terminal region on the activity and thermostability of orotate phosphoribosyltransferase (OPRTase, EC 2. 4.2.10) from Thermus thermophilus, four C-terminal amino acid-deleted OPRTases (1, 2, 3, and 5 residues deleted) were constructed. The activities of all the mutant OPRTases were lower than that of wild-type OPRTase at all temperatures investigated (50-80 degreesC). V- and EV-OPRTase, mutants with Val and Glu-Val deletions, respectively, showed 63 to 75% of the activity of wild-type OPRTase at the temperatures investigated. EEV- and PLEEV-OPRTase, with Glu-Glu-Val and Pro-Leu-Glu-Glu-Val deletions, respectively, had activities of 22 to 35% of the wild-type. The Km values for orotate of all mutant OPRTases were more than 4-fold higher than that of the wild-type (25 microM). On the other hand, the Km for PRPP of the wild-type was 34 microM, and there were no significant differences between the wild-type and mutant OPRTases. The kcat values of the V- and EV-OPRTases were similar to that of the wild-type, but those of the EEV- and PLEEV-OPRTases were less than 50% that of the wild-type. The optimum temperature of all mutant OPRTases, 70 degreesC, was 10 degreesC lower than that of the wild-type. The remaining activities of wild-type and V-OPRTase after incubation at 90 degreesC for 20 min were 70 and 60% of the non-treated OPRTase activity, respectively. Although the remaining activity of EV-OPRTase was only 14% of the non-treated OPRTase activity, the addition of 200 mM KCl during heat treatment increased it to 70%. Circular dichroism spectroscopy revealed that V- and EV-OPRTase denature more easily than the wild-type OPRTase. The results suggest that the C-terminal valine and glutamic acid residues are important for the activity and thermostability of T. thermophilus OPRTase.
Hamana
H
H
Department of Biological and Chemical Engineering, Faculty of Engineering, Gunma University, Kiryu, Gunma, 376-8515, Japan.
Shinozawa
T
T
D004795:Enzyme Stability; D006358:Hot Temperature; D007700:Kinetics; D009154:Mutation; D009962:Orotate Phosphoribosyltransferase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D009963:Orotic Acid / Q000378:metabolism; D010754:Phosphoribosyl Pyrophosphate / Q000378:metabolism; D011489:Protein Denaturation; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D017384:Sequence Deletion; D013696:Temperature; D016963:Thermus thermophilus / Q000201:enzymology*
D011994:Recombinant Proteins; D009963:Orotic Acid; D010754:Phosphoribosyl Pyrophosphate; D009962:Orotate Phosphoribosyltransferase