Highly conserved salt bridge stabilizes a proteinase K subfamily enzyme, Aqualysin I, from Thermus aquaticus YT-1.

Masayoshi Sakaguchi,Kanae Osaku,Masao Kawakita,Susumu Maejima,Nao Ohno,Fumitaka Oyama,Yasusato Sugahara

doi:10.1186/s13568-014-0059-2

Masayoshi Sakaguchi, Kanae Osaku + Show 5 more

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https://doi.org/10.1186/s13568-014-0059-2

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Abstract

The proteinase K subfamily enzymes, thermophilic Aqualysin I (AQN) from Thermus aquaticus YT-1 and psychrophilic serine protease (VPR) from Vibrio sp. PA-44, have six and seven salt bridges, respectively. To understand the possible significance of salt bridges in the thermal stability of AQN, we prepared mutant proteins in which amino acid residues participating in salt bridges common to proteinase K subfamily members and intrinsic to AQN were replaced to disrupt the bridges one at a time. Disruption of a salt bridge common to proteinase K subfamily enzymes in the D183N mutant resulted in a significant reduction in thermal stability, and a massive change in the content of the secondary structure was observed, even at 70°C, in the circular dichroism (CD) analysis. These results indicate that the common salt bridge Asp183-Arg12 is important in maintaining the conformation of proteinase K subfamily enzymes and suggest the importance of proximity between the regions around Asp183 and the N-terminal region around Arg12. Of the three mutants that lack an AQN intrinsic salt bridge, D212N was more prone to unfolding at 80°C than the wild-type enzyme. Similarly, D17N and E237Q were less thermostable than the wild-type enzyme, although this may be partially due to increased autolysis. The AQN intrinsic salt bridges appear to confer additional thermal stability to this enzyme. These findings will further our understanding of the factors involved in stabilizing protein structure.

Highlights

The molecular bases of protein adaptation to high and low temperatures are interesting from both basic and practical standpoints, as knowledge regarding these factors would enable the construction of genetically engineered proteins that could function under a variety of conditions
Based on an analysis of sequence homology, Aqualysin I (AQN) is classified into the proteinase K subfamily, which consists of a group of Gram-negative bacteria-derived proteinases within the subtilase superfamily (Siezen & Leunissen 1997)
These results are consistent with those reported for a subtilisin-like serine protease from Vibrio sp., VPR, which is a psychrophilic counterpart of AQN in the proteinase K subfamily (Kristjánsson et al 1999; Arnórsdóttir et al 2002)

Summary

Introduction

The molecular bases of protein adaptation to high and low temperatures are interesting from both basic and practical standpoints, as knowledge regarding these factors would enable the construction of genetically engineered proteins that could function under a variety of conditions. According to the MEROPS peptidase database (http:// merops.sanger.ac.uk/), subtilisin-like protease (subtilase) superfamily members are classified as the S8 subfamily in the serine protease superfamily These proteins exhibit a highly conserved arrangement of amino acids in the active site and have very similar overall structures consisting of an α/β protein scaffold. We demonstrated that Pro residues in the surface loops of AQN in the N-terminal region contribute significantly to its thermophilicity, and one of two disulfide bonds in AQN is more important for the catalytic activity and conformational stability of AQN than the other (Sakaguchi et al 2007; Sakaguchi et al 2008b) These results are consistent with those reported for a subtilisin-like serine protease from Vibrio sp., VPR, which is a psychrophilic counterpart of AQN in the proteinase K subfamily (Kristjánsson et al 1999; Arnórsdóttir et al 2002). It was found that the introduction of Pro residues into VPR at positions corresponding to those in AQN could improve its thermal stability (Arnórsdóttir et al 2009)

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