Cold adaption of enzymes: Structural comparison between salmon and bovine trypsins

Abstract

The crystal structure of an anionic form of salmon trypsin has been determined at 1.82 A resolution. We report the first structure of a trypsin from a phoikilothermic organism in a detailed comparison to mammalian trypsins in order to look for structural rationalizations for the cold-adaption features of salmon trypsin. This form of salmon trypsin (ST II) comprises 222 residues, and is homologous to bovine trypsin (BT) in about 65% of the primary structure. The tertiary structures are similar, with an overall displacement in main chain atomic positions between salmon trypsin and various crystal structures of bovine trypsin of about 0.8 A. Intramolecular hydrogen bonds and hydrophobic interactions are compared and discussed in order to estimate possible differences in molecular flexibility which might explain the higher catalytic efficiency and lower thermostability of salmon trypsin compared to bovine trypsin. No overall differences in intramolecular interactions are detected between the two structures, but there are differences in certain regions of the structures which may explain some of the observed differences in physical properties. The distribution of charged residues is different in the two trypsins, and the impact this might have on substrate affinity has been discussed.

Psychrophilic enzymes: molecular basis of cold adaptation

“…Although only one crystal structure from a psychrophilic enzyme is available ( [50], N. Aghajari and R. Haser, personal communication) in addition to the salmon trypsin [51] and elastase [52] structures, molecular modelling was extremely useful for preliminary structural analyses of cold-adapted enzymes. Structure prediction by homology modelling has recently reached sufficient level of reliability [53,54] to allow close inspection of the psychrophilic enzyme conformation, keeping in mind the limit of confidence of such models.…”

“…In the specific case of psychrophilic enzymes, there is however no direct experimental demonstration of such relationship. The crystallographic B-temperature factors (which are a function of individual atom mobility) of the recently crystallized h-amylase from the Antarctic psychrophile A. haloplanctis (N. Aghajari and R. Haser, personal communication) and of trypsin from the cold-adapted North Atlantic salmon [51] fail to reveal significant difference when compared with X-ray structures of mesophilic homologues. However, constraints imposed by the crystal packing of the molecules and intermolecular contacts can obscure possible differences occurring in solution.…”

“…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.…”

“…The individual contribution of Hbonds to protein stability is weak but the large number of these interactions in protein structures makes them a predominant factor of the folded state conformation [68]. The crystal structures of salmon and bovine trypsins [51] provided evidence that the disappearance of several H-bonds in the cold-adapted enzyme is responsible for loose contacts between the two i-barrel domains of the protein close to the catalytic residues, and between the C-terminal helix and the N-terminal domain. The total number of polar hydrogen-forming residues (Ser, Thr, Asn and Gln) is also lower in digestive enzymes from cold-adapted fish [69,70].…”

“…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.…”