Studies on structure and function of amine and amino acid related enzymes from microbiology.

从微生物学角度研究胺和氨基酸相关酶的结构和功能。

• 批准号: 06453168
• 负责人: KUMAGAI Hidehiko
• 金额: $ 4.74万
• 依托单位: KYOTO UNIVERSITY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 财政年份: 1994
• 资助国家: 日本
• 起止时间: 1994 至 1996
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-06453168/
• 关键词: gamma-glutamyltranspeptidase; monoamine oxidase; tyrosine phenol-lyase; Bifidobacterium; beta-glucosidase; amine oxidase; glutathione S-transferase; モノアミン酸化酵素; アミン酸化酵素; グルタチオンS-トランスフェラーゼ; X線結晶解析; γ-グルタミルトランスフェラーゼ

1. (1) Lys-257 of tyrosine phenol-lyase was mutated into Ala. And the strain over expresses the mutant enzyme was constructed using E.coli and T7 phage. (2) Transcription initiation site and regulation sites of tyrosine phenol-lyase gene was analyzed.2. (1) Structure of the main chain of gamma-glutamyltranspeptidase was determined. (2) Ser-33 of gamma-glutamyltranspeptidase was mutated into Cys. Pb^<2+> derivatives of the enzyme was analyzed. Mechanism of low temperature induction of the enzyme was analyzed. The induction was regulated by transcriptional regulation.3. (1) Monoamine oxidase of E.coli was induced by Cu^<2+> ion and the induction was regulated by transcriptional regulation. (2) The enzyme was induced by tyramine and also regulated by catabolite repression. (3) Over-expression of the regulator protein results in production of yellow enzyme. The yellow enzyme was purified and characterized. Its crystal structure was also studied.4.Copper-binding His residues and active center amino acid residues of amine oxidase of fungi were mutagenized.5.Isoenzyme gene of glutathione S-transferase was cloned from yeast. Over-expressed enzyme was purified and characterized.6.beta-Glucosidase gene of bifidobacterium was cloned in E.coli. Over-expressed enzyme was purified, characterized, and crystallized.

1. (1)将酪氨酸酚裂解酶的Lys-257突变为Ala。利用大肠杆菌和T7噬菌体构建了过量表达突变酶的菌株。(2)分析了酪氨酸酚裂合酶基因的转录起始位点和调控位点。(1)确定了γ-谷氨酰转肽酶的主链结构。(2)γ-谷氨酰转肽酶的第33位丝氨酸突变为半胱氨酸。分析了该酶的Pb^2+衍生物。对该酶的低温诱导机理进行了分析。诱导受转录调控. (1)大肠杆菌单胺氧化酶可被Cu^<2+>离子诱导，且诱导受转录调控。(2)该酶受酪胺诱导，也受分解代谢物阻遏的调节。(3)调节蛋白的过表达导致黄酶的产生。对黄酶进行了纯化和表征。4.对真菌胺氧化酶的铜结合组氨酸残基和活性中心氨基酸残基进行了诱变。5.从酵母中克隆了谷胱甘肽S-转移酶同工酶基因。6.在大肠杆菌中克隆了双歧杆菌β-葡萄糖苷酶基因。对过表达的酶进行纯化、表征和结晶。

项目成果

H.Suzuki.et al: "Escherichia coli k-12 can utilize an exogenous γ-glutamyl peptide as an amino acid source,for which γ-glutamyltranspeptidase is essential." J.Bacteriol.175. 6038-6040 (1993)

H.Suzuki.et al：“大肠杆菌 k-12 可以利用外源 γ-谷氨酰肽作为氨基酸来源，其中 γ-谷氨酰转肽酶是必需的。J.Bacteriol.175 (1993)。

N.Nunoura: "Purification and characterization of beta-D-glucosidase (beta-D-fucosidase) from Bifidobacteriumbreve clb acclimated to cellobiose" Biosci. Biotechnol. Biochem.60. 188-193 (1996)

N.Nunoura：“来自适应纤维二糖的短双歧杆菌 clb 的 β-D-葡萄糖苷酶（β-D-岩藻糖苷酶）的纯化和表征”Biosci。

Wataru Hahimoto: "Low temperature inducible γ-glutamyltranspeptidase of Escherichia coli K-12." Bioscience Biotechnology and Biochemistry. 61 (1). 34-39 (1996)

Wataru Hahimoto：“大肠杆菌 K-12 的低温诱导型 γ-谷氨酰转肽酶。”生物科学生物技术和生物化学 61 (1) (1996)。

Mitsuo Yamashita: "maoB,a gene that encodes a positive regulator of the monoamine oxidase gene (maoA) in Escherichia coli." Journal of Bacteriology. 178(10). 2941-2947 (1996)

Mitsuo Yamashita：“maoB，编码大肠杆菌中单胺氧化酶基因 (maoA) 正调节因子的基因。”

Hiroaki Sakai: "A nobel protein structure : γ-glutamyltranspeptidase from E. coli K-12." Journal of Biochemistry. 120 (1). 26-28 (1996)

Hiroaki Sakai：“一种诺贝尔蛋白质结构：来自大肠杆菌 K-12 的 γ-谷氨酰转肽酶”，《生物化学杂志》120 (1)。