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Published on: November 7, 2012
Q301P mutant of Vibrio PR protease affects activities under low-temperature and high-pressure conditions
Masahiko Okai1, Chiori Onoue2, Ryo Tsuda2
1Department of Ocean Sciences, Tokyo University of Marine Science and Technology, 4-5-7 Konan, Minato, Tokyo 108-8477, Japan.
Abstract:
We characterized a protease of the M4 family from the cold-adapted Vibrio sp. Pr21 that was isolated from seawater at 320-m deep in Sagami Bay, Japan, and named it as PR protease based on the strain name Pr21. The PR protease had activities at 10-60 °C and 0.1-350 MPa, with the optimal temperature and pressure at 40 °C and 250 MPa. The mutant 10C9 (Q301P) obtained by error-prone PCR had higher activities than the wild-type enzyme at 10-60 °C, and the Q301P mutation contributed to the increase of the activity. The specific activity value of 10C9 was also higher than that of the wild-type enzyme at 0.1-200 MPa, but the specific activity ratios (1.28-1.59) of 10C9/wild-type enzyme at 50-200 MPa at 30 °C were smaller than those at 10-60 °C (1.73-4.39) at 0.1 MPa. The catalytic efficiency value of 10C9 was lower than that of the wild-type enzyme at 200 MPa. The homology models of PR protease suggested that the side chain of Q301 was hydrogen-bonded with the carbonyl oxygen atom of the main chain of N234 in the wild-type enzyme, and P301 had no contact with N234 in 10C9. The break of the hydrogen bond in 10C9 might strengthen the increase of the flexibility of the β-sheet near the substrate binding pocket under high-temperature conditions, whereas the flexibility of the β-sheet in 10C9 might be moderately increased compared to that in the wild-type enzyme under high-pressure conditions.
Insights
We identified a novel M4 family protease, PR protease, from cold-adapted Vibrio sp. Pr21. A specific mutation (Q301P) enhanced its activity across a wide temperature and pressure range, indicating potential for industrial applications.
Area of Science:
- Biochemistry
- Enzymology
- Marine Microbiology
Background:
- The M4 family of proteases are crucial enzymes with diverse biological functions.
- Cold-adapted microorganisms, such as Vibrio sp. Pr21 isolated from deep seawater, possess unique enzymes adapted to extreme conditions.
- Understanding enzyme adaptations to high pressure and temperature is vital for biotechnology.
Purpose of the Study:
- To characterize a novel M4 family protease (PR protease) from the cold-adapted Vibrio sp. Pr21.
- To investigate the effects of a specific mutation (Q301P) on the enzyme's activity under varying temperature and pressure conditions.
- To elucidate the structural basis for the observed changes in enzyme activity.
Main Methods:
- Isolation and characterization of PR protease from Vibrio sp. Pr21.
- Enzyme activity assays across a range of temperatures (10–60 °C) and pressures (0.1–350 MPa).
- Site-directed mutagenesis (error-prone PCR) to generate the Q301P mutant (10C9).
- Homology modeling to predict structural changes.
Main Results:
- PR protease exhibited activity between 10–60 °C and 0.1–350 MPa, with optimal conditions at 40 °C and 250 MPa.
- The Q301P mutant (10C9) showed significantly higher activity than the wild-type enzyme across tested temperatures and pressures.
- Structural analysis suggested that breaking a hydrogen bond involving Q301 in the mutant enzyme increased flexibility, particularly under high-temperature conditions.
Conclusions:
- The PR protease from Vibrio sp. Pr21 is a robust enzyme with broad operational ranges.
- The Q301P mutation is key to enhancing the enzyme's stability and activity under challenging conditions.
- This study provides insights into enzyme adaptation mechanisms and the potential of deep-sea enzymes for biotechnological applications.
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