Stability and structural analysis of α‐amylase from the antarctic psychrophile Alteromonas haloplanctis A23

Abstract

The a-amylase secreted by the antarctic bacterium Alteromonas haloplanctis displays 66% amino acid sequence similarity with porcine pancreatic a-amylase. The psychrophilic a-amylase is however characterized by a sevenfold higher k,,, and k,,/K,,, values at 4°C and a lower conformational stability estimated as 10 kJ . mol-' 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 a-amylase, homology modelling of the psychrophilic a-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 @/a)* 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 C1F (Kd = 1.2 mM at 4°C) can result from single amino acid substitutions in the Ca2+-binding and CIF-binding sites and can also affect the compactness of a-amylase.

Psychrophilic enzymes: molecular basis of cold adaptation

“…In addition to the above mentioned crystal structures [50,51,52] models of psychrophilic enzymes such as bacterial subtilisin [59], h-amylase [45], triosephosphate isomerase [60], lipase [61], i-lactamase [48] or fish trypsin [62] and elastase [63] reveal that only subtle modifications of their conformation can be related to the low stability (see also [64,65]). Electrostatic weak interactions and the hydrophobic effect make a lower contribution to the global energy of stabilization of the native state whereas destabilizing forces mainly favour the conformational entropy of the unfolded state.…”

“…Among electrostatic weak interactions, ions pairs between two opposite side chain charges are the strongest stabilizing factors of protein conformation. When compared with mesophilic homologues, several psychrophilic enzymes lack surface salt bridges or ion pairs bonding secondary structures and protein domains [45,59]. Arginine residues also play a significant role in thermal adaptation.…”

“…In subtilisin, the low affinity seems to arise from the replacement of a strongly conserved polar ligand (Asn in mesophiles or thermophiles) by a threonine and the stacking of Gly residues around this ligand [59]. On the other hand, residues forming the calcium binding site of a psychrophilic h-amylase are identical to those of the mesophilic homologues: here, the low affinity for Ca 2 + probably results from the flexible structure of the cold-adapted enzyme [45]. Solvent interactions.…”

“…Attempts to evaluate this effect on the basis of structure have been performed, using the hydrophobic index of individual amino acids forming the protein core, or using hydrophobic cluster analysis. Numerous psychrophilic enzymes display substitutions within the hydrophobic core clusters that are accompanied by a sharp decrease of the hydrophobicity index when compared with mesophilic homologues [45,48,51,62]. Entropic factors.…”

Stepwise Adaptations to Low Temperature as Revealed by Multiple Mutants of Psychrophilic α-Amylase from Antarctic Bacterium

“…It displays striking sequence and structure similarities with its mesophilic homologue from pig pancreas (PPA) (11,(21)(22)(23). For instance, all 24 residues forming the catalytic cleft (supplemental Fig.…”