期刊
BIOCHIMICA ET BIOPHYSICA ACTA-PROTEINS AND PROTEOMICS
卷 1844, 期 6, 页码 1076-1082出版社
ELSEVIER SCIENCE BV
DOI: 10.1016/j.bbapap.2014.03.009
关键词
Cold-adapted enzyme; Esterase; Thermal stability; Catalytic site; Pseudomonas mandelii
资金
- Basic Science Research Program through the National Research Foundation of Korea (NRF) - Ministry of Education [NRF-2012R1A1A2042195]
Hydrophobic interactions are known to play an important role for cold-adaptation of proteins; however, the role of amino acid residue, Trp, has not been systematically investigated. The extracellular esterase, EstK, which was isolated from the cold-adapted bacterium Pseudomonas mandelii, has 5 Trp residues. In this study, the effects of Trp mutation on thermal stability, catalytic activity, and conformational change of EstK were investigated. Among the 5 Trp residues, W-208 was the most crucial in maintaining structural conformation and thermal stability of the enzyme. Surprisingly, mutation of W-208 to Tyr ((WY)-Y-208) showed an increased catalytic site thermal stability at ambient temperatures with a 13-fold increase in the activity at 40 degrees C compared to wild-type EstK. The structure model of (WY)-Y-208 suggested that Y-208 could form a hydrogen bond with D-308, which is located next to catalytic residue H-307, stabilizing the catalytic domain. Interestingly, Tyr was conserved in the corresponding position of hyper-thermophilic esterases EstE1 and AFEST, which are active at high temperatures. Our study provides a novel insight into the engineering of the catalytic site of cold-adapted enzymes with increased thermal stability and catalytic activity at ambient temperatures. (C) 2014 Elsevier B.V. All rights reserved.
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