Structure-Function of Novel Carboxyl Proteinases from Microorganisms

微生物新型羧基蛋白酶的结构-功能

• 批准号: 08044202
• 负责人: ODA Kohei
• 金额: $ 4.29万
• 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 international Scientific Research
• 财政年份: 1996
• 资助国家: 日本
• 起止时间: 1996 至 1998
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-08044202/
• 关键词: Prokaryote; Pepstatin; Carboxyl proteinase; Acid proteinase; Aspartic proteinase; Catalytic residue (s); Amino acid sequence; Structure-Function Relationship; カルボキシルプロテアーゼ; ペプスタチン

In this study, we aimed to identify the catalytic residues of pepstatin-insensitive carboxyl proteinases from prokaryote cells. We focussed 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. Substrate Specificity and Subsite Structure of KumamolysinSubstrate specificity of Kumamolysin was investigated by using two sets of synthetic substrates. The subsite structure of Kumamolysin was found to be different with those of aspartic proteinases. These data will be published in J.Biochem.2. Identification of Catalytic Residues by Using [^<14>C] … More Stylene OxideIn order to identify the catalytic residue(s) of XCP, [^<14>C] stylene oxide was used. It was found that the [^<14>C]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 [^<14>C] DCCDIn order to identify the catalytic residue(s) of PCP, chemical modification was carried out by using N.N'-dicyclohexylcafrbodiimide (DCCD) and specific inhibitor. tyrostatin, It was found that [^<14>C] 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 coagulansBacillus coagulans 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. 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）、黄单胞菌T-22（XCP）的羧基蛋白酶进行了研究。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，结构保守。在天冬氨酸蛋白酶活性中心没有观察到-Asp*-Thr-Gly-（Asp*：催化残基）。得到以下结果. Kumamolysin的底物特异性和亚位点结构通过使用两组合成底物研究Kumamolysin的底物特异性。Kumamolysin的亚位点结构与天冬氨酸蛋白酶不同。这些数据将发表在J.Biochem.2上。利用[^ C]鉴定催化残留物<14> ...更多信息 氧化苯乙烯为了鉴定XCP的催化残留物，使用了[13 <14>C]氧化苯乙烯。发现[^<14>C]环氧苯乙烯分别与XCP的E75和D110残基结合。其中，E75残基（对应于PCP的E80残基）及其邻近区域在PCP中较为保守，据此推测PCP的E80残基和XCP的E75残基分别作为底物结合位点参与了其催化功能.利用[^<14>C] DCCD鉴定五氯苯酚的催化残留物为了鉴定五氯苯酚的催化残留物，采用N，N '-二环己基咖啡二酰亚胺（DCCD）和特异性抑制剂对五氯苯酚进行化学修饰。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基因的克隆代号J-4。具有耐酒精和对胃蛋白酶抑制剂不敏感的特点。大部分基因已被克隆、测序。在获得全基因后，我们将尝试构建一个高效表达系统，并通过定点突变确定催化残基。少

项目成果

N.Oda,: "Nucleotide Sequence of the gene encoding the precursor protein of pepstatin-insensitive acid protease B, Scytali-dopepsin B, from Scytalidium lignicolum" Biosci.Biotech.Biochem.,. 62・8. 1637-1639 (1998)

N.Oda，：“编码来自Scytalidium lignicolum的胃酶抑素不敏感酸性蛋白酶B、Scytali-dopepsin B的前体蛋白的基因的核苷酸序列”Biosci.Biotech.Biochem.，1637-1639(1998)。

B.M.Dunn: "Aspartic Proteinases" M.James ed., Plenum Press, New York, 6 (1998)

B.M.Dunn：“天冬氨酸蛋白酶”M.James 编辑，Plenum Press，纽约，6 (1998)

K.Oda: "Xanthomonapepsin" Handbook of Proteolytic Enzymes (ed.A.J.Baret et al.). Academic Press. 2 (1998)

K.Oda：“Xanthomonapepsin”蛋白水解酶手册（ed.A.J.Baret 等人）。

K.Oda et al.: "Pepstatin-insensitive Carboxyl Proteinases from Prokaryotes Catalytic Residues and Substrate Specificities" Aspartic Proteinases (ed.by M.N.G.James). Plenum Press.New York. 5 (1998)

K.Oda 等人：“来自原核生物催化残基和底物特异性的胃酶抑素不敏感羧基蛋白酶”天冬氨酸蛋白酶（M.N.G.James 编辑）。

K.Oda,: "Cloning and expression of an isovaleryl pepstatin-insensi-tive carboxyl proteinase gene from Xanthomonas sp. T-22" J.Biochem.,. 120・3. 564-572 (1996)

K.Oda，：“来自黄单胞菌 T-22 的异戊酰胃酶抑素不敏感羧基蛋白酶基因的克隆和表达”J.Biochem.，120·3（1996）。