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，我们的目的是确定胃蛋白酶抑制剂不敏感的羧基蛋白酶的原核细胞的催化残基。本论文对假单胞菌101（PCP）的羧基蛋白酶进行了研究。黄单胞菌属T-22（XCP）、凝结芽孢杆菌J-4（J-4）和新芽孢杆菌（Bacillus novosp.）MN-32（阿莫溶血素）。它们的一级结构与迄今报道的天冬氨酸蛋白酶（胃蛋白酶抑制剂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）.利用定点突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）具有52%的同源性。基于高序列同一性，将催化残基（Asp或Glu）的8个氨基酸残基突出来，并且将它们全部突变为Ala残基。我们分析了这些 ...更多信息 具有自催化加工能力和蛋白酶活性的Ala突变体。因此，确定了PCP的一对D170和D328以及XCP的一对D169和D348为催化残基.用[14 C]氧化苯乙烯鉴别催化残留物为了鉴别XCP的催化残留物。[14 C]环氧乙烷。发现[14 C]环氧乙烷分别与XCP的E75和D110残基结合。其中，E75残基（对应于五氯苯酚的E80残基）及其邻近区域在五氯苯酚中是保守的。PCP的E80残基和XCP的E75残基分别作为底物结合位点参与其催化功能.利用[14 C]二环己基碳二亚胺（DCCD）和特异性抑制剂酪氨酸抑制素（tyrostatin）对五氯苯酚（PCP）进行化学修饰，以鉴定PCP的催化残留。结果表明，[14 C] 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）及其邻近区域在XCP中是保守的，并且E222 A突变体不具有蛋白酶活性，而XE 235 A（对应于PCP的E222 A）具有蛋白酶活性。基于这些数据。PCP的E222残基和XCP的E235残基被认为参与了它们的催化功能。可能分别作为底物结合位点。凝结芽孢杆菌羧基蛋白酶J-4基因的克隆命名为J-4，其特征在于耐酒精且对胃酶抑制剂不敏感。大部分基因已被克隆、测序。在获得整个基因后。我们将尝试构建一个高效表达系统，并通过定点突变来确定催化残基.新型芽孢杆菌Kumamolysin高效表达系统的构建MN-32羧基蛋白酶。命名为Kumamolysin。是一种耐热酶。我们成功地构建了该酶的高效表达系统。少

项目成果

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