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

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.