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

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，我们的目的是鉴定原核细胞中胃酶抑素不敏感的羧基蛋白酶的催化残基。我们的研究重点是假单胞菌的羧基蛋白酶。 101（PCP）。黄单胞菌属。 T-22 (XCP)、凝结芽孢杆菌 J-4 (J-4) 和芽孢杆菌。 MN-32（熊毛素溶解素）。它们的一级结构与迄今为止报道的天冬氨酸蛋白酶（胃酶抑素不敏感的羧基蛋白酶）没有任何相似之处。此外，在天冬氨酸蛋白酶活性中心没有观察到保守的结构-Asp*-Thr-Gly-(Asp*：催化残基)，得到以下结果： 1．使用定点诱变技术鉴定催化残基PCP（372个氨基酸残基）和XCP（398个氨基酸残基）彼此具有52%的同一性。基于高序列同一性，找出催化残基（Asp或Glu）的8个氨基酸残基，并将其全部突变为Ala残基。我们分析了这些 Ala 突变体的自催化加工能力和蛋白酶活性。结果，一对PCP的D170和D328以及XCP的一对D169和D348分别被鉴定为催化残基。 2．使用[14C]氧化苯乙烯鉴定催化残基为了鉴定XCP的催化残基，使用[14C]氧化苯乙烯。发现[14C]苯乙烯氧化物分别与XCP的E75和D110残基结合。其中，E75残基（对应于PCP的E80残基）及其附近在PCP中是保守的。基于这些数据。 PCP的E80残基和XCP的E75残基被认为分别作为底物结合位点参与其催化功能。 3．使用[14C]DCCD鉴定催化残基为了鉴定PCP的催化残基，使用N.N'-二环己基碳二亚胺(DCCD)和特异性抑制剂酪抑素进行化学修饰。发现[14C]DCCD分别与PCP的D140和E222残基结合。其中，E222残基（对应于XCP的E235残基）及其附近被发现在XCP中是保守的。此外，E222A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。 PCP的E222残基和XCP的E235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．来自凝结芽孢杆菌的羧基蛋白酶 J-4 基因的克隆凝结芽孢杆菌 J-4 羧基蛋白酶。指定为 J-4，其特点是具有酒精耐受性并且对胃酶抑素不敏感。大部分基因已被克隆、测序。得到完整基因后。我们将尝试构建高表达系统，并通过定点突变确定催化残基。 5．熊毛素溶血素高表达系统的构建。 MN-32 羧基蛋白酶。命名为 Kumamolysin。其特点是耐热酶。我们成功构建了该酶的高表达系统。较少的

项目成果

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