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Parvalbumins (PVs) from Antarctic notothenioid fishes display a pattern of thermal adaptation that likely reflects evolutionary changes in protein conformational flexibility. We have used ancestral sequence reconstruction and homology modeling to identify two amino acid changes that could potentially account for the present thermal sensitivity pattern of Antarctic fish PVs compared with a PV from a theoretical warm-adapted ancestral fish. To test this hypothesis, ancient PVs were resurrected in the lab using PV from the notothenioid Gobionotothen gibberifrons as a platform for introducing mutations comparable to the reconstructed ancestral PV sequences. The wild-type PV (WT) as well as three mutant expression constructs were engineered: lysine 8 to asparagine (K8N), lysine 26 to asparagine (K26N) and a double mutant (DM). Calcium equilibrium dissociation constants (K(d)) versus temperature curves for all mutants were right-shifted, as predicted, relative to that of WT PV. The K(d) values for the K8N and K26N single mutants were virtually identical at all temperatures and showed an intermediate level of thermal sensitivity. The DM construct displayed a full conversion of thermal sensitivity pattern to that of a PV from a warm/temperate-adapted fish. Additionally, the K(d) versus temperature curve for the WT construct revealed greater thermal sensitivity compared with the mutant constructs. Measurements of the rates of Ca(2+) dissociation (k(off)) showed that all mutants generally had slower k(off) values than WT at all temperatures. Calculated rates of Ca(2+) binding (k(on)) for the K8N and K26N mutants were similar to values for the WT PV at all temperatures. In contrast, the calculated k(on) values for the DM PV were faster, providing mechanistic insights into the nature of potentially adaptive changes in Ca(2+) binding in this PV. The overall results suggest that the current thermal phenotype of Antarctic PVs can be recapitulated by just two amino acid substitutions.

The uncharacterized α/β-hydrolase protein OLEI01171 from the psychrophilic marine bacterium Oleispira antarctica belongs to the PF00756 family of putative esterases, which also includes human esterase D. In the present paper we show that purified recombinant OLEI01171 exhibits high esterase activity against the model esterase substrate α-naphthyl acetate at 5-30°C with maximal activity at 15-20°C. The esterase activity of OLEI01171 was stimulated 3-8-fold by the addition of chloride or several other anions (0.1-1.0 M). Compared with mesophilic PF00756 esterases, OLEI01171 exhibited a lower overall protein thermostability. Two crystal structures of OLEI01171 were solved at 1.75 and 2.1 Å resolution and revealed a classical serine hydrolase catalytic triad and the presence of a chloride or bromide ion bound in the active site close to the catalytic Ser148. Both anions were found to co-ordinate a potential catalytic water molecule located in the vicinity of the catalytic triad His257. The results of the present study suggest that the bound anion perhaps contributes to the polarization of the catalytic water molecule and increases the rate of the hydrolysis of an acyl-enzyme intermediate. Alanine replacement mutagenesis of OLEI01171 identified ten amino acid residues important for esterase activity. The replacement of Asn225 by lysine had no significant effect on the activity or thermostability of OLEI01171, but resulted in a detectable increase of activity at 35-45°C. The present study has provided insight into the molecular mechanisms of activity of a cold-active and anion-activated carboxyl esterase.