title: Mutations in salt-bridging residues at the interface of the core and lid domains of epoxide hydrolase StEH1 affect regioselectivity, protein stability and hysteresis.
abstract: Epoxide hydrolase, StEH1, shows hysteretic behavior in the catalyzed hydrolysis of trans-2-methylstyrene oxide (2-MeSO)(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 alpha/beta-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 degrees C. Disruption of salt-bridges altered the rates for isomerization between distinct Michaelis complexes, with (1R,2R)-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.
authors.affiliation: Department of Biochemistry and Organic Chemistry, Uppsala University, Sweden.
authors.forename: Diana
authors.initials: D
authors.lastname: Lindberg
authors.forename: Shabbir
authors.initials: S
authors.lastname: Ahmad
authors.forename: Mikael
authors.initials: M
authors.lastname: Widersten
mesh_terms: D000595:Amino Acid Sequence; D004795:Enzyme Stability; D004851:Epoxide Hydrolases / Q000737:chemistry* / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D015870:Gene Expression; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010940:Plant Proteins / Q000737:chemistry* / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D012492:Salts / Q000737:chemistry*; D016415:Sequence Alignment; D011198:Solanum tuberosum / Q000201:enzymology*; D013379:Substrate Specificity; D013696:Temperature
chemical_list: D010940:Plant Proteins; D012492:Salts; D004851:Epoxide Hydrolases