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

在本研究中，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属 sp. 101 (PCP)、黄单胞菌属 sp. T-22 (XCP) 的羧基蛋白酶。 -4 (J-4) 和芽孢杆菌 MN-32。 (kumamolysin) 的主要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，并且 -Asp*-Thr-Gly-(Asp* :未观察到天冬氨酸蛋白酶活性中心的催化残基。得到如下结果： 1．Kumamolysin底物的底物特异性和亚位点结构。通过使用两组合成底物研究了 Kumamolysin 的亚位点结构，这些数据将发表在 J.Biochem.2 上。 14>C] … More 氧化苯乙烯为了鉴定 XCP 的催化残基，[^<14>C] 氧化苯乙烯被发现[^ 14 C]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.Based中是保守的。根据这些数据，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[^ 14 C]DCCD的催化残基为了鉴定PCP的催化残基，使用N，N'-二环己基cafrbodiimide(DCCD)和特异性抑制剂进行化学修饰，发现了。 [^ 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)及其附近被发现在XCP中是保守的。此外，根据这些数据，E222A突变体没有活性，而XE235A(对应于PCP的E222A)具有蛋白酶活性。 XCP的E235和E235被认为可能分别作为底物结合位点参与其催化功能。4．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的羧基蛋白酶的特征是具有酒精抗性并且对胃酶抑素不敏感，在获得完整基因之后，该酶被命名为J-4。我们将尝试构建高表达系统并通过定点突变来确定催化残基。

项目成果

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 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: "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）。

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)。

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）。