title: A single mutation Gln142Lys doubles the catalytic activity of VPR, a cold adapted subtilisin-like serine proteinase.
abstract: Structural comparisons of the cold adapted subtilase VPR and its thermophilic homologue, aqualysin I (AQUI) indicated the presence of additional salt bridges in the latter. Few of those appear to contribute significantly to thermal stability of AQUI. This includes a putative salt bridge between residues Lys142 and Glu172 as its deletion did not have any significant effect on its stability or activity (Jónsdóttir et al. (2014)). Insertion of this putative salt bridge into the structure of VPR, in a double mutant (VPRΔC_Q142K/S172E), however was detrimental to the stability of the enzyme. Incorporation of either the Q142K or S172E mutations into VPR, were found to significantly affect the catalytic properties of the enzyme. The single mutation Q142K was highly effective, as it increased the kcat and kcat/Km more than twofold. When the Q142K mutation was inserted into a thermostabilized, but a low activity mutant of VPR (VPRΔC_N3P/I5P), the activity increased about tenfold in terms of kcat and kcat/Km, while retaining the stability of the mutant. Molecular dynamics simulations of the single mutants were carried out to provide structural rationale for these experimental observations. Based on root mean square fluctuation (RMSF) profiles, the two mutants were more flexible in certain regions of the structure and the Q142K mutant had the highest overall flexibility of the three enzymes. The results suggest that weakening of specific H-bonds resulting from the mutations may be propagated over some distance giving rise to higher flexibility in the active site regions of the enzyme, causing higher catalytic activity in the mutants.
authors.affiliation: Department of Biochemistry, Science Institute, University of Iceland, Dunhagi 3, 107, Reykjavik, Iceland.
authors.forename: Kristinn R
authors.initials: KR
authors.lastname: Óskarsson
authors.affiliation: Computational Biology Laboratory, Unit of Statistics, Bioinformatics and Registry, Danish Cancer Society Research Center, Strandboulevarden 49, 2100, Copenhagen, Denmark.
authors.forename: Mads
authors.initials: M
authors.lastname: Nygaard
authors.affiliation: Department of Biochemistry, Science Institute, University of Iceland, Dunhagi 3, 107, Reykjavik, Iceland.
authors.forename: Brynjar Ö
authors.initials: BÖ
authors.lastname: Ellertsson
authors.affiliation: Institute of Biology, University of Iceland, Iceland.
authors.forename: Sigríður H
authors.initials: SH
authors.lastname: Thorbjarnardottir
authors.affiliation: Computational Biology Laboratory, Unit of Statistics, Bioinformatics and Registry, Danish Cancer Society Research Center, Strandboulevarden 49, 2100, Copenhagen, Denmark. Electronic address: elenap@cancer.dk.
authors.forename: Elena
authors.initials: E
authors.lastname: Papaleo
authors.affiliation: Department of Biochemistry, Science Institute, University of Iceland, Dunhagi 3, 107, Reykjavik, Iceland. Electronic address: mmk@hi.is.
authors.forename: Magnús M
authors.initials: MM
authors.lastname: Kristjánsson
mesh_terms: D000222:Adaptation, Physiological / Q000235:genetics; D002384:Catalysis; D020134:Catalytic Domain; D003080:Cold Temperature; D004795:Enzyme Stability / Q000235:genetics; D007700:Kinetics; D056004:Molecular Dynamics Simulation; D009154:Mutation / Q000235:genetics*; D018583:Pliability; D012697:Serine Endopeptidases / Q000235:genetics* / Q000378:metabolism*; D057057:Serine Proteases / Q000235:genetics* / Q000378:metabolism*; D012965:Sodium Chloride / Q000378:metabolism; D020860:Subtilisin / Q000235:genetics / Q000378:metabolism
chemical_list: D012965:Sodium Chloride; D057057:Serine Proteases; D012697:Serine Endopeptidases; C055102:aqualysin I; D020860:Subtilisin
keywords: Flexibility; Molecular dynamics; Protease; Subtilase; Temperature adaptation; Thermophilic