Structure-Function Relationships and Molecular Evolutions of Novel Carboxyl Proteinases from Microorganisms

微生物新型羧基蛋白酶的结构功能关系和分子进化

• 批准号: 09660089
• 负责人: ODA Kohei
• 金额: $ 2.05万
• 依托单位: Faculty of Textile Science, Kyoto Institute of Technology
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 1997
• 资助国家: 日本
• 起止时间: 1997 至 1998
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-09660089/
• 关键词: Prokaryote; Pepstatin; Carboxyl proteinase; Acid proteinase; Aspartic proteinase; Catalytic residue (s); Amino acid sequence; Structure-Function Relationship; 酵性プロテアーゼ; Prokaryote; Pepstatin; Carboxyl proteinase; Acid proteinase; Aspartic proteinase; Catalytic residue (s); Amino acid sequence; Structure-Function Relationship; 酵性プロテアーゼ

In this study, we aimed to identify the catalytic residues of pepstatin-insensitive carboxylproteinases from prokaryote cells. We focused our studies on carboxyl proteinases from Pseudomonas sp. 101 (PCP). Xanthomonas sp. T-22 (XCP), Bacillus coagulans J-4 (J-4), and Bacillus novosp. MN-32 (kumamolysin). The primary structures of them does not have any similarities to those of aspartic proteinases (pepstatin-insensitive carboxyl proteinase) reported so far. Moreover, the well-conserved structure, -Asp*-Thr-Gly-(Asp* : catalytic residue) in the active center of aspartic proteinases was not observed, The following results were obtained.1. Identification of Catalytic Residues by Using Site-directed Mutagenesis TechniquePCP (372 amino acid residues) and XCP (398 amino acid residues) have 52% identity to each other. Based on the high sequence identity, eight amino acid residues for catalytic residues (Asp or Glu) were poked up, and all of them were mutated to Ala residues. We analyzed these … More Ala mutants for both auto-catalytic processing ability and proteinase activity. Consequently, a pair of Dl70 and D328 for PCP.And a pair of D169 and D348 for XCP were identified as catalytic residues, respectively.2. Identification of Catalytic Residues by Using [14C] Stylene OxideIn order to identify the catalytic residue(s) of XCP.[14C]stylene oxide was used. It was found that the [14C]stylene oxide was bound to E75 and D110 residues of XCP, respectively. Of them, E75 residue (corresponding to E80 residue of PCP) and its vicinities were conserved in PCP.Based on these data. E80 residue of PCP and E75 residue of XCP were thought to be involved in their catalytic function, as a substrate binding site, respectively.3. Identification of Catalytic Residues by Using [14C] DCCDIn order to identify the catalytic residue(s) of PCP, chemical modification was carried out by using N.N'-dicyclohexylcarbodiimide (DCCD) and specific inhibitor, tyrostatin. It was found that [14C] DCCD was bound to D140 and E222 residues of PCP, respectively. Of them, E222 residue (corresponding to E235 residue of XCP) and its vicinities were found out to be conserved in XCP.Furthermore, E222A mutant had no any activity, whereas XE235A (corresponding to E222A for PCP) had proteinase activity. Based on these data. E222 residue of PCP and E235 of XCP were thought to be involved in their catalytic function. probably as a substrate binding site, respectively.4. Cloning of Carboxyl Proteinase J-4 Gene from Bacillus coagulansBacilluscoagulans J-4 carboxyl proteinase. designated as J-4, is characterized as alcohol resistant and insensitive to pepstatin. Most of the gene has been cloned, sequenced. After getting the whole gene. we will try to construct a high expression system and determine the catalytic residues by site-directed mutagenesis.5. Construction of High Expression System of KumamolysinBacillus novosp. MN-32 carboxyl proteinase. designated as Kumamolysin. is characterized as thermostable enzyme. We succeeded in constructing the high expression system for this enzyme. Less

在这项研究中，我们旨在从原核细胞中鉴定出对胃蛋白敏感的羧基蛋白酶的催化残留物。我们将研究重点放在假单胞菌sp的羧基蛋白酶上。 101（PCP）。 Xanthomonas sp。 T-22（XCP），柯瓜氏芽孢杆菌J-4（J-4）和Novosp芽孢杆菌。 MN-32（kumamolysin）。迄今为止，它们的主要结构与天冬氨酸蛋白酶（pepstatin不敏感的羧基蛋白酶）没有任何相似之处。此外，未观察到天冬氨酸蛋白酶活跃中心的保存良好的结构-asp* -thr-gly-（ASP*：催化居住），获得以下结果。1。通过使用位置定向的诱变技术（372个氨基酸保留）和XCP（398个氨基酸保留）鉴定催化残基，彼此具有52％的身份。基于高序列的身份，将催化残留物（ASP或GLU）的八个氨基酸残基戳戳，所有这些残留物被突变为ALA残基。我们分析了这些……更多的ALA突变体，用于自动催化加工能力和蛋白酶活性。因此，PCP的一对DL70和D328。 XCP的一对D169和D348分别鉴定为催化残留物。2。使用[14C]苯乙烯氧化物订单鉴定XCP的催化简历。[14C]氧化替代的催化恢复。发现[14C]氧化苯乙烯分别与XCP的E75和D110残差结合。其中，E75住宅（对应于PCP的E80居住）及其替代性在PCP中保存在这些数据上。 XCP的PCP和E75住所的E80住所分别与其催化功能有关，分别作为底物结合位点。3。使用[14C] DCCDIN顺序鉴定催化残基来鉴定PCP的催化保留率，通过使用N.N'-N'-dicyclohexylcarbodiimide（DCCD）和特定的抑制剂Trostatin进行化学修饰。发现[14C] DCCD分别与PCP的D140和E222残差结合。在其中，E222退休（对应于XCP的E235退休），并且发现其纤维率在XCP.Furthermore中配置为E2222a突变体没有任何活性，而XE235a（对应于PCP的E222a）具有蛋白酶活性。基于这些数据。 XCP的PCP和E235的E222残基被认为参与其催化功能。可能分别作为底物结合位点4。羧基蛋白酶J-4基因的克隆从豆科氏芽孢杆菌的基因J-4基因J-4 J-4羧基蛋白酶的克隆。被指定为J-4的特征是抗酒精性，对pepstatin不敏感。大多数基因已被克隆，测序。获得整个基因之后。我们将尝试构建一个高表达系统，并通过定点诱变确定催化性5。 Kumamolysinbacillus Novosp的高表达系统的建设。 MN-32羧基蛋白酶。被指定为kumamolysin。被称为热稳定酶。我们成功地为该酶构建了高表达系统。较少的