Mutations in salt-bridging residues at the interface of the core and lid domains of epoxide hydrolase StEH1 affect regioselectivity, protein stability and hysteresis

• Published in: Archives of biochemistry and biophysics Vol. 495; no. 2; pp. 165 - 173
• Main Authors: Lindberg, Diana, Ahmad, Shabbir, Widersten, Mikael
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 15.03.2010
• Subjects: Amino Acid Sequence; Biochemistry; Biokemi; Chemistry; Enzyme Stability; Epoxide hydrolase; Epoxide Hydrolases - chemistry; Epoxide Hydrolases - genetics; Epoxide Hydrolases - isolation & purification; Epoxide Hydrolases - metabolism; Gene Expression; Hysteresis; Kemi; Kinetics; Models, Molecular; Molecular Sequence Data; Mutagenesis; Mutagenesis, Site-Directed; NATURAL SCIENCES; NATURVETENSKAP; Plant Proteins - chemistry; Plant Proteins - genetics; Plant Proteins - isolation & purification; Plant Proteins - metabolism; Salt-bridges; Salts - chemistry; Sequence Alignment; Solanum tuberosum - enzymology; Substrate Specificity; Temperature; Mutagenesis; Epoxide hydrolase; Salt-bridges; Substrate specificity; Hysteresis
• ISSN: 0003-9861; 1096-0384; 1096-0384
• DOI: 10.1016/j.abb.2010.01.007

Epoxide hydrolase, StEH1, shows hysteretic behavior in the catalyzed hydrolysis of trans-2-methylstyrene oxide (2-MeSO) 1 Abbreviations used: SO, styrene oxide; 2-MeSO, trans-2-methylstyrene oxide. 1 . Linkage between protein structure dynamics and catalytic function was probed in mutant enzymes in which surface-located salt-bridging residues were substituted. Salt-bridges at the interface of the α/β-hydrolase fold core and lid domains, as well as between residues in the lid domain, between Lys 179-Asp 202, Glu 215-Arg 41 and Arg 236-Glu 165 were disrupted by mutations, K179Q, E215Q, R236K and R236Q. All mutants displayed enzyme activity with styrene oxide (SO) and 2-MeSO when assayed at 30 °C. Disruption of salt-bridges altered the rates for isomerization between distinct Michaelis complexes, with (1 R,2 R)-2-MeSO as substrate, presumably as a result of increased dynamics of involved protein segments. Another indication of increased flexibility was a lowered thermostability in all mutants. We propose that the alterations to regioselectivity in these mutants derive from an increased mobility in protein segments otherwise stabilized by salt bridging interactions.