Analysis and alteration of the substrate recognition machinery of stereospecific 2-hydroxyacid dehydrogenases from lactic acid bacteria.

乳酸菌立体特异性 2-羟基酸脱氢酶底物识别机制的分析和改变。

• 批准号: 15580067
• 负责人: TAGUCHI Hayao
• 金额: $ 2.11万
• 依托单位: Faculty of Science and Technology, Tokyo University of Science
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2003
• 资助国家: 日本
• 起止时间: 2003 至 2004
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-15580067/
• 关键词: Lactate dehydrogenase; Formate dehydrogenase; 2-Hydroxyacid dehydrogenase; Substrate specificity; Substrate recognition; Lactic acid bacteria; Allosteric enzyme; Catalysis; Lactobacillus; アロステリック酵素; タンパク質工学

For L.pentosus D-LDH, replacement of Tyr52 with Leu, Val, and Ala induced size-dependent changes in the specificity of the enzyme to aliphatic or aromatic 2-ketoacid substrates, indicating that the size or shape of hydrophobic side chain at position 52 determines the space of the binding site for hydrophobic side chains of 2-ketoacid substrates. The replacements of Tyr52 with Arg, Thr and Asp, and those of Phe299 with Gly and Ser greatly reduced the enzyme activities toward all 2-ketoacids tested, and induced slow but significant catalysis of NADH oxidation without substrate. However, the double mutations for positions 52 and 299 did not additively injured the enzyme function, but compensated each other for substrate-independent NADH oxidation and catalytic function for some substrates. Replacement of Asn97 with Asp did not markedly change the overall protein structure, but markedly perturbed the conformation of the active site loop in Lactobacillus pentosus D-LDH. The Asn97Asp mutant … More D-LDH exhibited virtually the same k_<cat>, but about 70-fold higher K_M value for pyruvate than the wild-type enzyme. For Paracoccus sp.12-A FDH, in contrast, replacement of Glu141 with Gln and Asn induced only 5.5- and 4.3-fold increases in the K_M value, but 110 and 590-fold decreases in the k_<cat> values for formate, respectively. Furthermore, these mutant FDHs, particularly the Glu141Asn enzyme, exhibited markedly enhanced catalytic activity for glyoxylate reduction, indicating that FDH is converted to a 2-hydroxyacid dehydrogenase on the replacement of Glu 141. These results indicate that the active site loops play different roles in the catalytic reactions of D-LDH and FDH-stabilization of substrate binding and promotion of hydrogen transfer, respectively-and that Asn97 and Glu141,which stabilize suitable loop conformations, are essential elements for proper loop functioning.For L.casei allosteric L-LDH, which exhibits relatively wide substrate specificity for 2-ketoacids, replacements of Arg173 with Gln and of His188 with Ala or Asp revealed that some 2-ketoacids can be bound to both the catalytic and allosteric sites of the enzyme, and the binding to the allosteric site can exhibit significant activation effects. These replacements also indicated that Arg173 is essential for the 2-ketoacid binding to the allosteric site, and His188 determines the specificity toward 2-ketoacids. Less

对于戊糖乳杆菌D-LDH，用Leu、瓦尔和Ala替换Tyr 52诱导酶对脂肪族或芳香族2-酮酸底物的特异性的大小依赖性变化，表明位置52处的疏水侧链的大小或形状决定2-酮酸底物的疏水侧链的结合位点的空间。用Arg、Thr和Asp取代Tyr 52，用Gly和Ser取代Phe 299，大大降低了对所有2-酮酸的酶活性，并诱导了缓慢但显著的无底物NADH氧化催化。然而，位置52和299的双突变并没有加性地损害酶的功能，但相互补偿底物非依赖性的NADH氧化和催化功能的一些底物。天冬氨酸取代Asn 97没有显着改变整体蛋白质结构，但显着扰乱了戊糖乳杆菌D-LDH中的活性位点环的构象。Asn 97 Asp突变体 ...更多信息 D-LDH<cat>对丙酮酸的K_M值比野生型酶高70倍。而对于副球菌12-A FDH，Gln和Asn分别取代Glu 141后，其K_M值仅增加5.5和4.3倍，而甲酸盐的K_M值分别降低110和590倍<cat>。此外，这些突变FDH，特别是Glu 141 Asn酶，表现出显着增强的催化活性乙醛酸还原，表明FDH被转化为2-羟基酸脱氢酶的Glu 141的替代。这些结果表明，活性中心环在D-LDH和FDH的催化反应中分别起着稳定底物结合和促进氢转移的不同作用，而Asn 97和Glu 141稳定合适的环构象，是适当环功能的必要元素。对于干酪乳杆菌变构L-LDH，它对2-酮酸具有相对广泛的底物特异性，用Gln取代Arg 173和用Ala或Asp取代His 188表明，一些2-酮酸可以结合到酶的催化位点和变构位点上，并且结合到变构位点上可以表现出显著的激活效应。这些替换也表明，Arg 173是必不可少的2-酮酸结合变构位点，和His 188决定对2-酮酸的特异性。少

项目成果

Distinct conformation-mediated functions of an active site loop in the catalytic reactions and formate dehydrogenase of NAD-dependent D-lactate dehydrogenase

NAD依赖性D-乳酸脱氢酶的催化反应和甲酸脱氢酶中活性位点环的独特构象介导功能

• 发表时间: 2005
• 期刊: J.Biol.Chem. 280
• 作者: 篠田 剛

タンパク質工学の基礎

蛋白质工程基础

• 发表时间: 2004
• 作者: 篠田 剛;Takeshi Shinoda;徳田千束;Chizuka Tokuda;田口速男 他9名;松澤 洋
• 通讯作者: 松澤 洋

Conversion of Lactobacillus pentosus D-lactate dehydrogenase to a D-hydroxyisocaproate dehydrogenase through a single amino acid replacement

• DOI: 10.1128/jb.185.16.5023-5026.2003
• 发表时间: 2003-08-01
• 期刊: JOURNAL OF BACTERIOLOGY
• 影响因子: 3.2
• 作者: Tokuda, C;Ishikura, Y;Taguchi, H
• 通讯作者: Taguchi, H

田口速男 他10名: "Conversion of Lactobacillus pentosus D-lactate dehydrogenase to a D-hydroxyisocaproate dehydrogenase through a single amino acid replacement."Journal of Bacteriology. 185・16. 5023-5026 (2003)

Hayao Taguchi 等 10 人：“通过单一氨基酸替换将戊糖乳杆菌 D-乳酸脱氢酶转化为 D-羟基异己酸脱氢酶”。细菌学杂志 185・16（2003 年）。

Distinct Conformation-mediated Functions of an Active Site Loop in the Catalytic Reactions of NAD-dependent D-Lactate Dehydrogenase and Formate Dehydrogenase*

• DOI: 10.1074/jbc.m500970200
• 发表时间: 2005-04
• 期刊: Journal of Biological Chemistry
• 影响因子: 4.8
• 作者: Takeshi Shinoda;Kazuhito Arai;Mayu Shigematsu-Iida;Yoshirou Ishikura;Satoru Tanaka;Takashi Yamada;M. Kimber;E. Pai;S. Fushinobu;H. Taguchi