Department of Material and Life Science, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka 565-0871, Japan.
Protein Eng Des Sel. 2011 May;24(5):411-8. doi: 10.1093/protein/gzq122. Epub 2011 Jan 7.
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.
一个位于假单胞菌 MIS38 家族 I.3 脂肪酶（PML）C 末端 15-19 位的五残基序列基序（VTLVG）和位于 PML C 末端的极端 C 末端基序（DGIVIA）在 1 型分泌系统（T1SS）的载体蛋白中相对保守。为了分析这些基序的作用，构建了四个 PML 突变体蛋白（PMLΔ5、PMLΔ10、3A-PML 和 2A-PML）。PMLΔ5 和 PMLΔ10 分别缺失了 PML 的 C 末端 5 和 10 个残基。3A-PML 在极端 C 末端基序内有三个突变，2A-PML 在五残基序列基序内有两个突变。使用携带 Lip 系统（I.3 脂肪酶的 T1SS）的大肠杆菌 DH5 细胞分析这些蛋白质的分泌。与 PML 相比，2A-PML 的分泌水平显著降低，而 3A-PML 的分泌水平与 PML 相当，表明五残基序列基序而不是极端 C 末端基序是 PML 分泌所必需的。这些突变和缺失都没有严重影响 PML 的酶活性。然而，3A-PML、PMLΔ5 和 PMLΔ10 的 T(1/2)分别比 PML 低 2.1、7.6 和 7.6°C，ΔG(H(2)O)分别低 5.0、21.3 和 17.9 kJ/mol。这些结果表明，PML 的极端 C 末端基序对稳定性很重要。
