Enhanced catalytic site thermal stability of cold-adapted esterase EstK by a W208Y mutation

Abstract

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, W208 was the most crucial in maintaining structural conformation and thermal stability of the enzyme. Surprisingly, mutation of W208 to Tyr (W208Y) showed an increased catalytic site thermal stability at ambient temperatures with a 13-fold increase in the activity at 40°C compared to wild-type EstK. The structure model of W208Y suggested that Y208 could form a hydrogen bond with D308, which is located next to catalytic residue H307, 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.