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

在这项研究中，我们旨在从原核细胞中鉴定胃抑素不敏感的羧化蛋白酶的催化残基。我们主要研究假单胞菌101 （Pseudomonas sp. 101， PCP）的羧基蛋白酶。黄单胞菌T-22 （XCP）、凝固芽孢杆菌J-4 （J-4）和新芽孢杆菌。MN-32 (kumamolysin)。它们的一级结构与目前报道的天冬氨酸蛋白酶（胃抑素不敏感的羧基蛋白酶）没有任何相似之处。此外，在天冬氨酸蛋白酶的活性中心未观察到保守性较好的-Asp*- thr - gly -（Asp*：催化残基）结构。利用位点诱变技术鉴定催化残基epcp（372个氨基酸残基）和XCP（398个氨基酸残基）的同源性为52%。基于高序列同源性，挖掘出8个催化残基（Asp或Glu），全部突变为Ala残基。我们分析了这些突变体的自催化处理能力和蛋白酶活性。因此，一对Dl70和D328用于PCP。XCP的D169和D348分别为催化残基。用[14C]苯乙烯氧化法鉴定XCP的催化残留物。[14C]环氧乙烷。发现[14C]环氧乙烷分别与XCP的E75和D110残基结合。其中E75残基（对应PCP的E80残基）及其邻区在PCP中较为保守。基于这些数据。PCP的E80残基和XCP的E75残基分别作为底物结合位点参与了它们的催化作用。为了鉴定PCP的催化残留物，采用n - n′-双环己基碳二亚胺（DCCD）和特异性抑制剂酪氨酸抑素对其进行了化学修饰。发现[14C] DCCD分别与PCP的D140和E222残基结合。其中E222残基（对应XCP的E235残基）及其邻区在XCP中被发现是保守的。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残基被认为参与了它们的催化作用。可能分别作为底物结合位点。凝固芽孢杆菌J-4羧基蛋白酶基因的克隆被命名为J-4，其特点是耐酒精，对胃抑素不敏感。大部分基因已经被克隆，测序。在获得整个基因之后。我们将尝试构建一个高表达系统，并通过位点定向诱变确定催化残基。熊果溶菌素新芽孢杆菌高表达体系的构建。MN-32羧基蛋白酶。被命名为熊熊素。是一种耐热酶。我们成功构建了该酶的高表达体系。少

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