Journal of Biochemistry Advance Access originally published online on December 14, 2006
Journal of Biochemistry 2007 141(2):213-220; doi:10.1093/jb/mvm025
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© 2006 The Japanese Biochemical Society.
Role of Proline Residues in Conferring Thermostability on Aqualysin I
Department of Applied Chemistry, Kogakuin University, 1-24-2 Nishishinjuku, Shinjuku-ku, Tokyo 163-8677, Japan
*To whom correspondence should be addressed. Tel: +81-3-3340-2731, Fax: +81-3-3340-0147, E-mail: bt13004{at}ns.kogakuin.ac.jp
Received October 31, 2006; Accepted December 2, 2006
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Abstract |
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To understand the molecular basis of the thermostability of a thermophilic serine protease aqualysin I from Thermus aquaticus YT-1, we introduced mutations at Pro5, Pro7, Pro240 and Pro268, which are located on the surface loops of aqualysin I, by changing these amino acid residues into those found at the corresponding locations in VPR, a psychrophilic serine protease from Vibrio sp. PA-44. All mutants were expressed stably and exhibited essentially the same specific activity as wild-type aqualysin I at 40°C. The P240N mutant protein had similar thermostability to wild-type aqualysin I, but P5N and P268T showed lower thermostability, with a half-life at 90°C of 15 and 30 min, respectively, as compared to 45 min for the wild-type enzyme. The thermostability of P7I was decreased even more markedly, and the mutant protein was rapidly inactivated at 80°C and even at 70°C, with half-lives of 10 and 60 min, respectively. Differential scanning calorimetry analysis showed that the transition temperatures of wild-type enzyme, P5N, P7I, P240N and P268T were 93.99°C, 83.45°C, 75.66°C, 91.78°C and 86.49°C, respectively. These results underscore the importance of the proline residues in the N- and C-terminal regions of aqualysin I in maintaining the integrity of the overall protein structure at elevated temperatures.
Key Words: proline, serine protease, subtilase, thermostability
Abbreviations: AAPF, N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide; DSC, differential scanning calorimetry; IPTG, isopropyl ß-D-thiogalactopyranoside; LB, Luria-Bertani; MES, 2-(N-morphlino)ethane sulfonic acid; MOE, The Molecular Operating Environment; ODA, oligonucleotide-directed dual amber; PMSF, phenylmethanesulfonyl fluoride; TCA, trichloroacetic acid; VPR, proteinase from Vibrio PA-44
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