Stability and structural analysis of α‐amylase from the antarctic psychrophile <i>Alteromonas haloplanctis</i> A23

Abstract

The alpha-amylase secreted by the antarctic bacterium Alteromonas haloplanctis displays 66% amino acid sequence similarity with porcine pancreatic alpha-amylase. The psychrophilic alpha-amylase is however characterized by a sevenfold higher kcat and kcat/Km values at 4 degrees C and a lower conformational stability estimated as 10 kJ.mol-1 with respect to the porcine enzyme. It is proposed that both properties arise from an increase in molecular flexibility required to compensate for the reduction of reaction rates at low temperatures. This is supported by the fast denaturation rates induced by temperature, urea or guanidinium chloride and by the shift towards low temperatures of the apparent optimal temperature of activity. When compared with the known three-dimensional structure of porcine pancreatic alpha-amylase, homology modelling of the psychrophilic alpha-amylase reveals several features which may be assumed to be responsible for a more flexible, heat-labile conformation: the lack of several surface salt bridges in the (beta/alpha)8 domain, the reduction of the number of weakly polar interactions involving an aromatic side chain, a lower hydrophobicity associated with the increased flexibility index of amino acids forming the hydrophobic clusters and by substitutions of proline for alanine residues in loops connecting secondary structures. The weaker affinity of the enzyme for Ca2+ (Kd = 44 nM) and for Cl- (Kd = 1.2 mM at 4 degrees C) can result from single amino acid substitutions in the Ca(2+)-binding and Cl(-)-binding sites and can also affect the compactness of alpha-amylase.