Effects of Metal Ions on Stability and Activity of Hyperthermophilic Pyrolysin and Further Stabilization of This Enzyme by Modification of a Ca ²⁺ -Binding Site

Abstract

Pyrolysin is an extracellular subtilase produced by the marine hyperthermophilic archaeon Pyrococcus furiosus. This enzyme functions at high temperatures in seawater, but little is known about the effects of metal ions on the properties of pyrolysin. Here, we report that the supplementation of Na ؉ , Ca 2؉ , or Mg 2؉ salts at concentrations similar to those in seawater destabilizes recombinant pyrolysin but leads to an increase in enzyme activity. The destabilizing effect of metal ions on pyrolysin appears to be related to the disturbance of surface electrostatic interactions of the enzyme. In addition, mutational analysis of two predicted high-affinity Ca 2؉ -binding sites (Ca1 and Ca2) revealed that the binding of Ca 2؉ is important for the stabilization of this enzyme. Interestingly, Asn substitutions at residues Asp818 and Asp820 of the Ca2 site, which is located in the C-terminal extension of pyrolysin, resulted in improvements in both enzyme thermostability and activity without affecting Ca 2؉ -binding affinity. These effects were most likely due to the elimination of unfavorable electrostatic repulsion at the Ca2 site. Together, these results suggest that metal ions play important roles in modulating the stability and activity of pyrolysin. Hyperthermophiles grow optimally at temperatures of 80°C or higher (1), and some can survive and reproduce at temperatures higher than 120°C (2, 3). Enzymes from hyperthermophiles are especially valuable for probing the stabilization mechanisms that allow proteins to function near the maximum temperature of life, and studying these enzymes also offers insight into opportunities to greatly expand the reaction conditions of biocatalysis (4-6). At high temperatures, inactivation of enzymes occurs mainly due to thermal denaturation (loss of tertiary structure) and degradation (loss of primary structure) (7). Accumulating evidence suggests that hyperthermophilic enzymes have evolved multiple mechanisms to adapt to hyperthermal environments; however, no general rules for stabilization of these enzymes have been described (5,8). Known intrinsic stabilization factors include improved hydrophobic and aromatic interactions, increased ionic interactions, decreased entropy of the unfolded state and solvent-accessible hydrophobic surface, anchoring of loose ends, intersubunit interactions and oligomerization, metal binding, and posttranslational modifications (5, 8-11). Certain hyperthermophiles possess a specific protein disulfide oxidoreductase and use disulfide bonding as a major mechanism for protein stabilization (12, 13). In addition to the intrinsic factors, some hyperthermophilic enzymes are stabilized by extrinsic factors, such as intracellular compatible solutes and molecular chaperones, ligand binding, crowding effects inside the cells, and association with the surface layer (S-layer) (5,8,14,15). Although hyperthermophilic enzymes have become model systems to investigate mechanisms of enzyme stabilization and have provided the rational basis for improving the stability ...

Four Inserts within the Catalytic Domain Confer Extra Stability and Activity to Hyperthermostable Pyrolysin from Pyrococcus furiosus

“…Instead, Arg 249 appears to form unfavorable electrostatic interactions that destabilize pyrolysin. Our previous study showed that Asn substitutions at the Ca2 site residues Asp 818 and Asp 820 in pyrolysin enhances the thermostability of the enzyme (15). When incubated at 95°C, the variants R249E and D818N/D820N had a similar half-life of approximately 18 h, which is longer than that of the WT (approximately 12 h) (Fig.…”

“…Salt-induced autocleavage within IS29 and IS27 produces nicked pyrolysin. We showed previously that pyrolysin undergoes autocleavage when supplemented with various salts (15). Here, the cleavage sites were identified by N-terminal sequencing.…”

“…6A). Md was previously identified as an mPls degradation product produced by C-terminal truncation (15). Md exhibited proteolytic activity in the absence of trichloroacetic acid (TCA) treatment and was degraded and inactivated in the presence of salt (Fig.…”

“…Interestingly, the Ca1 site also contains a DFTDE sequence involved in Ca 2ϩ binding ( Fig. 2) (15). The Dx[DN]xDG motifs and their downstream sequences are highly conserved in several pyrolysin-like proteases (Fig.…”

“…We previously identified two calcium-binding sites (Ca1 and Ca2) in pyrolysin (15). Sequence analysis of IS147 revealed two Dx[DN]xDG motifs, Asp 132 -Gly 137 (motif 1) and Asp 161 -Tyr 166 (motif 2) (Fig.…”