ANALYSIS OF SUBSTRATE RECOGNITION MECHANISM OF ISOPROPYLMALATE DEHYDROGENASE

苹果酸异丙酯脱氢酶的底物识别机制分析

• 批准号: 10680612
• 负责人: TANAKA Hidehiko
• 金额: $ 1.86万
• 依托单位: OKAYAMA UNIVERSITY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 1998
• 资助国家: 日本
• 起止时间: 1998 至 2000
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-10680612/
• 关键词: ISOPROPYLMALATE DEHYDROGENASE; ISOCITRATE DEHYDROGENASE; SUBSTRATE RECOGNITION MECHANISM; COENZYME SPECIFICITY; X-RAY CRYSTALLOGRAPHY; 基質認識部位; 変異導入プライマー; イソクエン酸・脱水素酵素; 3-イソプロピルリン丁酸脱水素酵素

1) Structure of 3-isopropylmalate dehydrogenase from Thiobacillus ferrooxidans at 2.0 Åresolution.3-Isopropylmalate dehydrogenase (IPMDH) gene was cloned and sequenced from Thiobacillus ferrooxidans (Tf). We have determined the crystal structures of Tf-IPMDH and the substrate binding mechanism have been solved. IPMDH shows a marked similarity to isocitrate dehydrogenase (ICDH) in its structural framework and catalytic mechanism, and is classified into a unique group of decarboxylating dehydrogenases. The difference between their substrates is the γ moiety attached to 2R-malate.The structure shows a fully closed conformation and the substrate-binding site is quite similar to that of ICDH except for a region around the γ-isopropyl group. The γgroup is recognized by a unique hydrophobic pocket, which includes Glu88, Leu91 and Leu92. The Cβ and Cγ atoms of Glu88 interact with the γ-isopropyl moiety of 3-isopropylmalate (IPM) and are central to the recognition of substrate.2) Mutagenesis of … More substrate recognition site of Tf-IPMDH.We have changed γmoiety recognition site of Tf-IPMDH (Ala72〜Leu92) to that of Ec-ICDH (Pro102〜Val116). We analyzed the substrate specificity of the mutant enzyme. We have been investigated whether the higher-order structure for the mutant enzyme is stabilized because about ten amino acid residues of leuB gene are substituted. In result, The mutant enzyme's loop regions will be distorted slightly, but we expected the structure of the mutant enzyme was stabilized and began on this theme. The mutant enzyme was expressed as soluble protein in E.coli.3 )Structure of NAD dependent ICDH from Thiobacillus thiooxidans and coenzyme recognition mechanism.NAD dependent ICDH gene from Thiobacillus thiooxidans (Tt) was cloned and sequenced and the coenzyme recognition mechanism was analyzed. The amino acid sequence had a high degree of similarity to the NADP dependent ICDH and IPMDH.Tt-ICDH is 59.2%, and 22.6% identical to Ec-ICDH and Tf-IPMDH, respectively. It is expected that the tertiary structure of Tt-ICDH is similar to Ec-ICDH and Tf-IPMDH, because the regions composed a deduced secondary structure (α-helix and β-sheet) of Tt-ICDH are much alike.Tt-ICDH has conserved Asp357 which is significant of the NAD recognition in the coenzyme binding site alignment. On the other hand, Tt-ICDH lacked for a tyrosine residue which was conserved in NADP-ICDH and recognized phosphate of NADP+. Thus, it is revealed that this enzyme has similar coenzyme binding site to that of the IPMDH's. Less

1)氧化亚铁硫杆菌3-异丙基苹果酸脱氢酶（IPMDH）基因的克隆及序列分析。我们测定了Tf-IPMDH的晶体结构，并解决了底物结合机理。IPMDH与异柠檬酸脱氢酶（ICDH）在结构和催化机制上有很大的相似性，属于一类独特的脱羧酶。它们的底物不同之处在于γ部分连接在2 R-苹果酸上，其结构显示完全闭合的构象，底物结合位点与ICDH非常相似，除了γ-异丙基周围的区域。γ基团由独特的疏水口袋识别，包括Glu 88，Leu 91和Leu 92。Glu 88的Cβ和Cγ原子与3-异丙基苹果酸（IPM）的γ-异丙基部分相互作用，并且是识别底物的中心。 ...更多信息 Tf-IPMDH的底物识别位点：我们将Tf-IPMDH的γ部分识别位点（Ala 72 → Leu 92）改为Ec-ICDH的γ部分识别位点（Pro 102 → Val 116）。我们分析了突变酶的底物特异性。我们已经研究了突变酶的高级结构是否稳定，因为leuB基因的大约10个氨基酸残基被取代。结果，突变酶的环区会有轻微的变形，但我们预期突变酶的结构是稳定的，并以此为主题开始。3）氧化硫硫杆菌NAD依赖型ICDH的结构及辅酶识别机制对氧化硫硫杆菌（Tt）NAD依赖型ICDH基因进行了克隆和测序，并对其辅酶识别机制进行了分析。该氨基酸序列与NADP依赖的ICDH和IPMDH具有高度相似性，Tt-ICDH与Ec-ICDH和Tf-IPMDH的相似性分别为59.2%和22.6%。Tt-ICDH的二级结构（α-螺旋和β-折叠）与Ec-ICDH和Tf-IPMDH相似，Tt-ICDH具有保守的Asp 357结构，Asp 357在辅酶结合位点比对中对NAD的识别具有重要意义。另一方面，Tt-ICDH缺乏NADP-ICDH中保守的酪氨酸残基，并识别NADP+的磷酸。因此，这表明该酶具有与IPMDH类似的辅酶结合位点。少

项目成果

Seow Teck Keong, Kenji Inagaki, T.Nakamura, R.Maeda, Takashi Tamura, and Hidehiko Tanaka: "Purification and Some Characteristics of a Monomeric Alanine Racemase from an Extreme thermophile, Thermus thermophilus"J.Biosci.Bioeng.. 90. 344-346 (2000)

Seow Teck Keong、Kenji Inagaki、T.Nakamura、R.Maeda、Takashi Tamura 和 Hidehiko Tanaka：“来自极端嗜热菌、嗜热栖热菌的单体丙氨酸消旋酶的纯化和一些特征”J.Biosci.Bioeng.. 90. 344

Inoue,H.,K.Inagaki,N.Adachi,T.Tamura,N.Esaki,K.Soda and H.Tanaka: "Role of Tyrosine 114 of L-Methionine γ-Lyase from Pseudomonas putida"Biosci.Biotech.Biochem. 64・11. 2332-2339 (2000)

Inoue, H.、K. Inagaki、N. Adachi、T. Tamura、N. Esaki、K. Soda 和 H. Tanaka：“来自恶臭假单胞菌的 L-蛋氨酸 γ-裂解酶的酪氨酸 114 的作用”Biosci.Biotech.Biochem 64·11。2332-2339（2000）

Tamura,T.,Y.Oki,A.Yoshida,T.Kuriyama,H.Kawakami,H.Inoue,K.Inagaki and H.Tanaka: "Noncompetitive, reversible inhibition of aminoacylase-l by a series of l-α-hydroxyl-and l-α-fluoro-fatty acids; the ligand specificity of Aspergillus oryzae and porcine kidne

Tamura, T., Y. Oki, A. Yoshida, T. Kuriyama, H. Kawakami, H. Inoue, K. Inagaki 和 H. Tanaka：“一系列 l-α- 对氨酰化酶-l 的非竞争性、可逆抑制羟基-和l-α-氟代脂肪酸；米曲霉和猪肾的配体特异性

Ohhira,I.,T.Tamura,H.Kurokawa,H.Nakaue,K.Inagaki,and H.Tanaka: "Purification of anti-Escherichia coli O-157 components produced by Enterococcus faecalis TH10, an isolate from Malaysian fermentation food, Tempeh"Milk Science. 49・2. 81-88 (2000)

Ohhira, I.、T. Tamura、H. Kurokawa、H. Nakaue、K. Inagaki 和 H. Tanaka：“粪肠球菌 TH10（一种从马来西亚发酵食品中分离出来的菌株）产生的抗大肠杆菌 O-157 成分的纯化，豆豉《乳科学》. 49・2. 81-88 (2000)

Tamura,T.,Y.Oki,A.Yoshida,T.Kuriyama,H.Kawakami,H.Inoue,K.Inagaki and H.Tanaka: "Noncompetitive, reversible inhibition of aminoacylase-1 by a series of l-α-hydroxyl-and l-α-fluoro-fatty acids; the ligand specificity of Aspergillus oryzae and porcine kidne

Tamura, T., Y. Oki, A. Yoshida, T. Kuriyama, H. Kawakami, H. Inoue, K. Inagaki 和 H. Tanaka：“一系列 l-α- 对aminoacylase-1 的非竞争性、可逆抑制羟基-和l-α-氟代脂肪酸；米曲霉和猪肾的配体特异性