A novel cold-adapted esterase FE2495 from Antarctic Flavobacterium ardleyense A2-1: Biochemical characterization and structural basis for cold-adaptation.

Abstract

Cold-adapted enzymes from microorganisms living in polar zones represent promising biocatalysts for low-temperature applications. In this study, we identified a novel esterase, FE2495, from the Antarctic bacterium Flavobacterium ardleyense A2-1 and investigated underlying mechanisms for its cold-adaptive properties. The enzyme featured a canonical catalytic triad (Ser113, Asp167, His198), with the nucleophilic Ser113 residing within a conserved GFSQG motif. Phylogenetic analysis classified FE2495 as a family VI esterase. Nearly 50% of the enzyme's esterase activity was retained at temperatures ranging from 0 to 10 °C, and near maximal activity (80%) observed at 25-45 °C. The FE2495 enzyme displayed a marked preference for hydrolyzing short-chain acyl esters, with highest activity toward p-nitrophenyl acetate (C2) and diminishing activity toward longer-chain substrates (C2 > C4 = C6 > C8 > C10). The enzyme also exhibited remarkable halotolerance and distinct stereoselectivity toward chiral esters. Esterase activity of FE2495 was enhanced by K+ and Mg2+ but strongly inhibited by the transition metals Zn2+ and Cu2+. Comparative structural analysis with a thermophilic homolog Est5250 revealed that FE2495 possessed a large catalytic pocket, fewer hydrogen bonds and salt bridges, and a distinct amino acid composition depleted in arginine, providing a possible explanation for its broad temperature tolerance. A V169Y substitution near the catalytic Asp167 prevented activity toward longer acyl-chain substrates, while allowing the hydrolysis of pNP(C2) and specific stereoisomers. The V169Y substitution also reduced Km and impaired catalytic efficiency toward pNP(C2) by stabilizing a non-productive enzyme-substrate complex.