Porin bearing the protease activity : its structure and function

具有蛋白酶活性的孔蛋白：其结构和功能

• 批准号: 07670327
• 负责人: YOSHIHARA Eisaku
• 金额: $ 1.6万
• 依托单位: Tokai University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 1995
• 资助国家: 日本
• 起止时间: 1995 至 1996
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-07670327/
• 关键词: Pseudomonas aeruginosa; outer membrane protein; porin; protein D2; protease; site-directed mutagenesis; catalytic residue; mutifunctional protein; protein D2; 外膜; ptoteinD2; チャンネル蛋白; 塩基性アミノ酸; モノクローナル抗体

We found out that protein D2 (OprD) porin in the outer membrane of Pseudomonas aeruginosa bears the protease activity. This was concluded from the following results. (a) Highly purified OprD sample hydrolyzed the synthetic peptides according to the Michaelis-Menten kinetics, (b), diisopropyl fluorophosphate (DFP), an inhibitor specific for serine protease, inhibited the hydrolytic activity and bound covalently with OprD protein, and (c), monoclonal antibody raised against OprD protein inhibited the protease activity in a concentration-dependent manner. However, one may suspect that the protease activity may be mediated by very small amounts of protease (s) in the OprD preparation. Therefore, in order to demonstrate conclusively the protease activity in OprD protein and simultaniouly identfy the catalytic residues of OprD protease, we constructed the mutant OprD proteins with the substitution of His156, Asp208 or Ser296 with glutamine, asparagine or alanine, respectively since these amino acids were predicted to be catalytic triad constituents from the comparison analysisi of the amino acid sequence between OprD and serine protease such as trypsin. The wild-type and mutant OprD proteins expressed in the outer membrane of these strains were purified to be homogeneity and were subjected to the protease assay. Consequently all the three mutants were shown to have the protease activity with less than 0.1% of the wild-type OprD activity. This result demonstrates conclusively that OprD protein itselu has the protease and that His156, Asp208 and Ser296 residues are the constituents of the catalytic triad of OprD protease. Furthermore, we studied the role of these catalytic residues to the interaction with imipenem, one of carbapenems since the OprD was shown to have the binding site to imipenem. Susceptiblities of these mutant strains agains imipem showed that His156 and Asp208 but not Ser296 may have some role for the fasilitaed diffusion of imipem through OprD protein.

我们发现铜绿假单胞菌外膜的蛋白D2（OprD）孔蛋白具有蛋白酶活性。这是根据以下结果得出的结论。 (a) 高度纯化的 OprD 样品根据 Michaelis-Menten 动力学水解合成肽，(b) 氟磷酸二异丙酯 (DFP)，一种丝氨酸蛋白酶特异性抑制剂，抑制水解活性并与 OprD 蛋白共价结合，(c) ），针对 OprD 蛋白的单克隆抗体以浓度依赖性方式抑制蛋白酶活性。然而，人们可能怀疑蛋白酶活性可能是由 OprD 制剂中极少量的蛋白酶介导的。因此，为了最终证明OprD蛋白的蛋白酶活性，同时鉴定OprD蛋白酶的催化残基，我们构建了突变型OprD蛋白，其中His156、Asp208或Ser296分别被谷氨酰胺、天冬酰胺或丙氨酸取代，因为这些氨基酸通过对OprD和丝氨酸之间的氨基酸序列的比较分析，预测它们是催化三联体成分蛋白酶，例如胰蛋白酶。将这些菌株外膜表达的野生型和突变型OprD蛋白纯化至同质并进行蛋白酶测定。因此，所有三个突变体都显示出具有小于野生型OprD活性0.1%的蛋白酶活性。该结果最终证明OprD蛋白本身具有蛋白酶并且His156、Asp208和Ser296残基是OprD蛋白酶的催化三联体的成分。此外，我们研究了这些催化残基在与亚胺培南（碳青霉烯类之一）相互作用中的作用，因为OprD被证明具有与亚胺培南的结合位点。这些突变菌株对亚胺培平的敏感性表明，His156 和 Asp208 而不是 Ser296 可能对亚胺培平通过 OprD 蛋白的扩散有一定作用。

项目成果

Nakae, T., Yoshihara, E., Yoneyama, H.And Ishii J.: "Protein D2, a multifuctional channel-forming outer membrane protein of Pseudomonas aeruginosa" Molecular Biology of Pseudomonads (Nakazawa et al.Eds). 363-370 (1996)

Nakae, T.、Yoshihara, E.、Yoneyama, H. 和 Ishii J.：“蛋白质 D2，铜绿假单胞菌的多功能通道形成外膜蛋白”假单胞菌分子生物学（Nakazawa 等编辑）。

Yoshihara, E., Gotoh, N., Nishino, T.And Nakae, T.: "Protein D2 porin of the Pseudomonas aeruginosa outer membrane bears the protease activity" FEBS Letters. 394. 179-182 (1996)

Yoshihara, E.、Gotoh, N.、Nishino, T. 和 Nakae, T.：“铜绿假单胞菌外膜的蛋白质 D2 孔蛋白具有蛋白酶活性”FEBS 快报。

Nakae,Yoshihara et al.: "Protein D2,a multifunctional channel-forming outer membrane protein of Pseudomonas aeruginosa" Molecular Biology of Pseudomonads(Nakazawa et al.Eds). 363-370 (1996)

Nakae，Yoshihara 等人：“蛋白质 D2，铜绿假单胞菌的多功能通道形成外膜蛋白”假单胞菌的分子生物学（Nakazawa 等人编辑）。

Nakae,Yoshihara,et al.: "Protein D2,a multifunctional channel-forming outer membrane protein of Pseudomonas aeruginosa" Molecular Biology of Pseudomonads (Nakazawa et al. Eds). 363-370 (1996)

Nakae，Yoshihara 等人：“蛋白质 D2，铜绿假单胞菌的多功能通道形成外膜蛋白”假单胞菌的分子生物学（Nakazawa 等人编辑）。

Yoshihara,E,et al.: "Protein D2 porin of the Pseudomonas aeruginosa outer membrane bears the protease activity" FEBS Letters. 394. 179-182 (1996)

Yoshihara,E,et al.：“铜绿假单胞菌外膜的蛋白质 D2 孔蛋白具有蛋白酶活性”FEBS Letters。