title: Importance of an extreme C-terminal motif of a family I.3 lipase for stability.
abstract: A five-residue sequence motif (VTLVG) located at positions 15-19 from the C-terminus of family I.3 lipase from Pseudomonas sp. MIS38 (PML) and an extreme C-terminal motif (DGIVIA) located at the C-terminus of PML are relatively well conserved in the passenger proteins of type 1 secretion system (T1SS). To analyze the role of these motifs, four mutant proteins of PML (PMLΔ5, PMLΔ10, 3A-PML and 2A-PML) were constructed. PMLΔ5 and PMLΔ10 lack the C-terminal 5 and 10 residues of PML, respectively. 3A-PML has triple mutations within an extreme C-terminal motif and 2A-PML has double mutations within a five-residue sequence motif. Secretion of these proteins was analyzed using Escherichia coli DH5 cells carrying Lip system (T1SS for family I.3 lipase). The secretion level of 2A-PML was dramatically reduced when compared with that of PML, whereas the secretion level of 3A-PML was comparable to that of PML, indicating that a five-residue sequence motif, instead of an extreme C-terminal motif, is required for secretion of PML. None of the mutations and truncations seriously affects the enzymatic activity of PML. However, 3A-PML, PMLΔ5 and PMLΔ10 were less stable than PML by 2.1, 7.6 and 7.6°C in T(1/2), respectively, and by 5.0, 21.3 and 17.9 kJ/mol in ΔG(H(2)O), respectively. These results indicate that an extreme C-terminal motif of PML is important for stability.
authors.affiliation: Department of Material and Life Science, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka 565-0871, Japan.
authors.forename: K
authors.initials: K
authors.lastname: Kuwahara
authors.forename: C
authors.initials: C
authors.lastname: Angkawidjaja
authors.forename: Y
authors.initials: Y
authors.lastname: Koga
authors.forename: K
authors.initials: K
authors.lastname: Takano
authors.forename: S
authors.initials: S
authors.lastname: Kanaya
mesh_terms: D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D004795:Enzyme Stability; D004926:Escherichia coli / Q000166:cytology / Q000235:genetics; D008049:Lipase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011489:Protein Denaturation / Q000187:drug effects; D011549:Pseudomonas / Q000201:enzymology*; D017384:Sequence Deletion; D013696:Temperature; D014508:Urea / Q000494:pharmacology
chemical_list: D014508:Urea; D008049:Lipase