PuSH - Publication Server of Helmholtz Zentrum München

Sigurdardottir, A.G.* ; Arnórsdóttir, J.* ; Thorbjarnardóttir, S.H.* ; Eggertsson, G.* ; Suhre, K. ; Kristjánsson, M.M.*

Characteristics of mutants designed to incorporate a new ion pair into the structure of a cold adapted subtilisin-like serine proteinase.

Biochim. Biophys. Acta-Proteins Proteom. 1794, 512-518 (2009)
DOI PMC
Open Access Green as soon as Postprint is submitted to ZB.
Structural comparisons of VPR, a subtilisin-like serine proteinase from a psychrotrophic Vibrio species and a thermophilic homologue, aqualysin 1. have led us to hypothesize about the roles of different residues in the temperature adaptation of the enzymes. Some of these hypotheses are now being examined by analysis of mutants of the enzymes. The selected substitutions are believed to increase the stability of the cold adapted enzyme based on structural analysis of the thermostable structure. We report here on mutants, which were designed to incorporate an ion pair into the structure of VPR. The residues Asp17 and Arg259 are assumed to form an ion pair in aqualysin 1. The cold adapted VPR contains Asn (Asn15) and Lys (Lys257) at corresponding sites in its structure. In VPR. Asn 15 is located on a surface loop with its side group pointing towards the side chain of Lys257. By substituting Asn15 by Asp (N15D) it was considered feasible that a salt bridge would form between the oppositely charged groups. To mimic further the putative salt bridge from the thermophile enzyme the corresponding double mutant (N15D/K257R) was also produced. The N15D mutation increased the thermal stability of VPR by similar to 3 degrees C, both in T-50% and T-m. Addition of the K257R mutation did not however, increase the stability of the double mutant any further. Despite this stabilization of the VPR mutants the catalytic activity (k(cat)) against the substrate Suc-AAPF-NH-Np was increased in the mutants. Molecular dynamics simulations on wild type and the two mutant proteins suggested that indeed a salt bridge was formed in both cases. Furthermore, a truncated form of the N15D mutant (N15D Delta C) was produced, lacking a 15 residue long C-terminal extended sequence not present in the thermophilic enzyme. In wild type VPR this supposedly moveable, negatively charged arm on the protein molecule might interfere with the new salt bridge introduced as a result of the N15D mutation. Removal of the C-terminal arm improved the thermal stability (T-m similar to+1.5 degrees C) of the truncated enzyme (VPR Delta C) as compared to the wild type VPR. Introduction of the N15D substitution into VPR Delta C improved the thermal stability further by about 3 degrees C, or to about the same extent as in the wild-type. However, contrary to what was observed for the wild type, the introduction of the putative salt bridge did not affect the catalytic properties (k(cat)) of the C-terminal truncated enzyme.
Altmetric
Additional Metrics?
[➜Log in]
Edit extra informations Login
Publication type Article: Journal article
Document type Scientific Article
Corresponding Author
Keywords Ion pair; Stability; Kinetic property; Site directed mutagenesis; Cold adaptation; Subtilisin-like; Vibrio-proteinase; salmon salmo-salar; cod gadus-morhua; crystal-structure; aqualysin-i; hyperthermophilic proteins; charge interactions; adenylate kinases; citrate synthase; adaptation; stability
ISSN (print) / ISBN 1570-9639
e-ISSN 1878-1454
Journal Biochimica et Biophysica Acta - Proteins and Proteomics
Quellenangaben Volume: 1794, Issue: 3, Pages: 512-518 Article Number: , Supplement: ,
Publisher Elsevier
Non-patent literature Publications
Reviewing status Peer reviewed
Institute(s) Institute of Bioinformatics and Systems Biology (IBIS)