Reaction Mechanisms and Application of New Bacterial Enzymes

新型细菌酶的反应机理及应用

• 批准号: 08044218
• 负责人: ASANO Yasuhisa
• 金额: $ 6.27万
• 依托单位: Toyama Prefectural University
• 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 1997
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-08044218/
• 关键词: Amino Acid Dehydrogenase; Opine Dehydrogenase; X-ray Crystallographic Studies; Site Directed Mutagenesis; Molecular Cloning; Leucine Dehydrogenase; Phenylalanine Dehydrogenase; Methylaspartate Ammonia-Lyase; X線構造解析; ロイシン脱水素酵素

(1) Phenylalanine dehydrogenase (PheDH) : PheDHs from Bacillus sphaericus, B.badius, Sporosarcina ureae were purified from Escherichia coli transformants in large scales and sent to Prof.Engel (University College Dublin, Ireland) and Prof.Rice (University of Sheffield, U.K.) for X-ray crystallography. Based on informations of X-ray crystallography of glutamate dehydrogenase, chimeric enzymes of PheDH with altered substrate specificities were constructed.(2) Methylaspartate ammonia-lyase (MAL) : MALs from Enterobacteria such as Enterobacter, Citrobacter, Proteus were purified to homogeneities and their enxymological properties were analyzed in detail. MAL from Citrobacter amalonaticus strain YG-1002 was digested with endo peptidases and N-terminal amino acid sequences were determined. Based on the information, DNA probes were synthesized. The gene for the enzyme was cloned form the genomic library of c.amalonaticus strain YG-1002 by PCR and Southern hybridization. The sequence of the gene was compared with that of a strict anaerobe Clostridium tetanomorphum. MAL was crystallized and sent to Prof.Rice for studies of X-ray crystallography.(3) Opine dehydrogenase (ODH) : The gene for ODH from Arthrobacter sp.IC was cloned and expressed in E.coli. The enzyme was purified in a large scale and sent to Prof.Rice, and on its X-ray studies were started. The first structure of a (D,L) superfamily member, N-(1-D-Carboxylethyl)-L-norvaline dehydrogenase from Arthrobacter sp.strain 1C,has been solved to 1.8* resolution and the location of the bound coenzyme determined.(4) Results were presented in some international academic meetings.

(1)苯丙氨酸脱氢酶（PheDH）：从大肠杆菌转化体中大规模纯化来自球形芽孢杆菌（Bacillussphaericus）、巴氏芽孢杆菌（B.badius）、铜绿八叠球菌（Sporosarcinaueruginosa）的PheDH，并送至Engel教授（都柏林大学学院，爱尔兰）和Rice教授（英国谢菲尔德大学）.用于X射线晶体学。基于谷氨酸脱氢酶的X射线晶体学信息，构建了底物特异性改变的PheDH嵌合酶。(2)甲基天冬氨酸解氨酶（MAL）：从肠杆菌属、柠檬酸杆菌属、变形杆菌属等肠杆菌中纯化MAL，并对其酶学性质进行了详细分析。用内切肽酶消化来自柠檬酸杆菌YG-1002菌株的MAL，并测定N-末端氨基酸序列。根据这些信息，合成了DNA探针。利用PCR和Southern杂交技术，从大肠杆菌YG-1002菌株基因组文库中克隆了该酶的基因。将该基因的序列与严格厌氧菌破伤风梭菌的序列进行了比较。MAL被结晶化，并被送到赖斯教授那里进行X射线晶体学研究。(3)冠瘿碱脱氢酶（ODH）：克隆了节杆菌IC的ODH基因，并在大肠杆菌中表达。这种酶被大规模纯化并送到赖斯教授那里，并开始了X射线研究。节杆菌属菌株1C的（D，L）超家族成员N-（1-D-羧乙基）-L-正缬氨酸脱氢酶的第一个结构已解析到1.8 ×分辨率，并确定了结合辅酶的位置。(4)在一些国际学术会议上介绍了成果。

项目成果

K.L. Britton: "Crystallisation of Arthrobacter sp.strain 1C opine dehydrogenase and its complex with NAD^+." Acta Crystallography D. 54. 124-126 (1998)

K.L.

K.L.Britton: "Crystallisation of Arthrobacter sp.strain 1C opine dehydrogenase and its complex with NAD+." Acta Crystallography D. (in press).

K.L.Britton：“节杆菌属菌株 1C 鸦片脱氢酶及其与 NAD 复合物的结晶。”

Stephen Y.K.Seah: "Alteration in relative activities of phenylalanine dehydrogenase towards different substrates by site-directed mutagenesis." FEBS Letters. 370. 93-96 (1995)

Stephen Y.K.Seah：“通过定点诱变改变苯丙氨酸脱氢酶对不同底物的相对活性。”

K.Nakamura: "Quantitation of L-amino acids by cycling between an aminotransferase and a dehydrogenase : application to the determination of L-phenylalanine in human serum" Analytical Biochemistry. 234. 19-22 (1996)

K.Nakamura：“通过转氨酶和脱氢酶之间的循环定量 L-氨基酸：应用于测定人血清中的 L-苯丙氨酸”分析生物化学。

Y.Kato: "Stereoselective synthesis of opine-type secondary amine carboxylic acids by a new enzyme opine dehydrogenase. -Use of recombinant enzymes-" Journal of Molecular Catalysis B: Enzymatic. 1. 151-160 (1996)

Y.Kato：“通过新的欧品脱氢酶立体选择性合成欧品型仲胺羧酸。-重组酶的使用-”《分子催化杂志 B：酶学》。