title: Calcium-independent opening of lid1 of a family I.3 lipase by a single Asp to Arg mutation at the calcium-binding site.
abstract: A family I.3 lipase from Pseudomonas sp. MIS38 (PML) has two lids, lid1 and lid2, which are open when it exhibits activity. A single calcium ion is required to anchor lid1 in the open conformation by coordination with two acidic residues (Asp153 and Asp157) in lid1 and three other residues. Lid1 adopts a long α-helix in the open conformation, whereas it is sharply bent within this helix, such that Asp153 and Asp157 are distantly located to each other, in the closed conformation. To examine whether the mutation of Asp153 or Asp157 to a positively charged residue allows two residues at Positions 153 and 157 to come close with each other and thereby stabilizes the open conformation of lid1 even in the absence of calcium ions, five single mutant proteins (D153K-, D153R-, D153A-, D157K- and D157R-PMLs) and two double mutant proteins (D153A/D157A- and D153R/D157N-PMLs) were constructed. Of these mutant proteins, only D153R-PML exhibited activity in the absence of calcium ions. Its lipase and esterase activities were 7-fold lower and 4-fold higher than those of PML, respectively. These activities were lost by the mutation of Asp157 to Asn. These results suggest that lid1 of D153R-PML opens even in the absence of calcium ions due to electrostatic attraction between Arg153 and Asp157.
authors.affiliation: Department of Material and Life Science, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka 565-0871, Japan.
authors.forename: M
authors.initials: M
authors.lastname: Cheng
authors.forename: C
authors.initials: C
authors.lastname: Angkawidjaja
authors.forename: Y
authors.initials: Y
authors.lastname: Koga
authors.forename: S
authors.initials: S
authors.lastname: Kanaya
mesh_terms: D019943:Amino Acid Substitution; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism*; D004789:Enzyme Activation; D008049:Lipase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D009154:Mutation; D011487:Protein Conformation; D015202:Protein Engineering / Q000379:methods*; D011549:Pseudomonas / Q000201:enzymology*; D055672:Static Electricity; D013379:Substrate Specificity
chemical_list: D008049:Lipase; D002118:Calcium
keywords: Pseudomonas; calcium-binding site; lid; lipase; open conformation