Department of Material and Life Science, Graduate School of Engineering, Osaka University, Japan.
FEBS J. 2012 Mar;279(6):976-86. doi: 10.1111/j.1742-4658.2012.08483.x. Epub 2012 Feb 6.
FKBP22 from the psychotropic bacterium Shewanella sp. SIB1 is a homodimeric protein with peptidyl prolyl cis-trans isomerase (PPIase) activity. According to a tertiary model, several nonpolar residues including Trp157 and Phe197 form a substrate-binding cavity, and Asp137 and Arg142, which form a salt bridge, are located at the edge of this cavity. To analyze the role of these residues, nine single (D137A, R142A, W157A/F/Y, F197A/L/Y/W) and one double (D137A/R142A) mutant protein of SIB1 FKBP22 were constructed. The far- and near-UV CD spectra of these mutant proteins suggest that the mutations at Asp137 and Arg142 do not seriously affect the protein structure, while those at Trp157 and Phe197 cause a local conformational change around the mutation site. Each mutation decreased the PPIase activities of SIB1 FKBP22 for peptide and protein substrates similarly without seriously affecting chaperone function. This result indicates that SIB1 FKBP22 does not require PPIase activity for chaperone function. The PPIase activities of R142A, D137A and D137A/R142A decreased in this order, suggesting that Asp137 and Arg142 play a principal and auxiliary role in catalytic function, respectively, but Arg142 can function as a substitute of Asp137. Because the PPIase activity of SIB1 FKBP22 was not fully lost by the removal of all polar residues around the active site, the desolvation effect may also contribute to the enzymatic activity. However, the mutations of Trp157 to Phe or Phe197 to Leu greatly decrease the enzymatic activity, suggesting that the shape of the substrate-binding cavity is also important for enzymatic activity.
来自精神药物细菌希瓦氏菌 SIB1 的 FKBP22 是一种具有肽基脯氨酰顺反异构酶 (PPIase) 活性的同二聚体蛋白。根据三级模型，包括色氨酸 157 和苯丙氨酸 197 在内的几个非极性残基形成一个底物结合腔，位于该腔边缘的天冬氨酸 137 和精氨酸 142 形成盐桥。为了分析这些残基的作用，构建了 SIB1 FKBP22 的 9 个单（D137A、R142A、W157A/F/Y、F197A/L/Y/W）和 1 个双（D137A/R142A）突变体蛋白。这些突变蛋白的远紫外和近紫外圆二色谱表明，天冬氨酸 137 和精氨酸 142 的突变不会严重影响蛋白质结构，而色氨酸 157 和苯丙氨酸 197 的突变则导致突变部位周围的局部构象变化。每个突变都降低了 SIB1 FKBP22 对肽和蛋白质底物的 PPIase 活性，而没有严重影响伴侣功能。结果表明，SIB1 FKBP22 不需要 PPIase 活性来发挥伴侣功能。R142A、D137A 和 D137A/R142A 的 PPIase 活性依次降低，表明天冬氨酸 137 和精氨酸 142 分别在催化功能中起主要和辅助作用，但精氨酸 142 可以作为天冬氨酸 137 的替代物。由于去除活性位点周围所有极性残基并未完全丧失 SIB1 FKBP22 的 PPIase 活性，去溶剂化效应也可能有助于酶活性。然而，色氨酸 157 突变为苯丙氨酸或苯丙氨酸 197 突变为亮氨酸会大大降低酶活性，表明底物结合腔的形状对酶活性也很重要。
