Molecular and cellular studies on biofunctional proteins from microorganisms.

微生物生物功能蛋白的分子和细胞研究。

• 批准号: 10306007
• 负责人: KUMAGAI Hidehiko
• 金额: $ 21.25万
• 依托单位: KYOTO UNIVERSITY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (A).
• 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-10306007/
• 关键词: glycopeptide; glycosidase; transglycosidation; glutathione; aminopeptidase; γ-glutamyltranspeptidase; cAMP; pseudohyphal growth; チロシンフェノールリアーゼ; グルコース受容体; チラミン酸化酵素; 大腸菌; ペプチダーゼ; モノアミン酸化酵素; 出芽酵母; レセプター; 結晶構造解析

1. We analyzed the function of specific glycosidases produced by microorganisms and attempted to produce useful substances using these enzymes. (a) Enzymatic syntheses of various bioactive glycopeptides were carried out using transglycosylation activity of microbial glycosidases. (b) Effect of glycosylation on the function of protein was investigated using microbial glycosidases.2. We found that aminopeptidases A, B and N, and dipeptidase D with broad substrate specificity are the four cysteinylglycinases of Escherichia coli K-12. Enzymatic characteristics of purified aminopeptidase B were determined.3. We identified that the catalytic nucleophile of Escherichia coli γ-glutamyltranspeptidase is the Thrresidue at the N-terminal of the small subunit by a new novel affinity labeling agent.4. We found that Gpr1, a putative G-protein coupled receptor, regulated glucose dependent cellular cAMP level in yeast Saccharomyces cerevisiae. Functional analysis revealed that pseudohyphal development observed under high glucose and low nitrogen condition was reduced in the GPR1 gene disruptant. We also clarified that the reduced transcriptional level of FLO11 gene which encodes cell surface protein involved in cell adhesion caused pseudohyphal defect in the GPR1 disruptant.

1.我们分析了由微生物产生的特异性糖苷酶的功能，并尝试使用这些酶生产有用的物质。(a)利用微生物糖苷酶的转糖基化活性进行各种生物活性糖肽的酶促合成。(b)利用微生物糖苷酶研究了糖基化对蛋白质功能的影响.我们发现，氨肽酶A，B和N，和具有广泛的底物特异性的二肽酶D是大肠杆菌K-12的四个半胱氨酰甘氨酸酶。对纯化的氨肽酶B进行了酶学性质测定。通过一种新的亲和标记试剂，确定大肠杆菌γ-谷氨酰转肽酶的催化亲核体为小亚基N端的Thr残基.我们发现，Gpr 1，一个假定的G-蛋白偶联受体，调节葡萄糖依赖的细胞cAMP水平在酵母酿酒酵母。功能分析表明，在高糖低氮条件下观察到的假菌丝发育减少GPR 1基因破坏剂。我们还阐明了在GPR 1破坏剂中，编码参与细胞粘附的细胞表面蛋白的FLO 11基因的转录水平降低导致假菌丝缺陷。

项目成果

Tamaki H.et al.: "GPRI regulates filamentous growth through FLOII in yeast Saccharomyces cerevisiae"Biochem. Biophys. Res. Commun.. 267. 164-168 (2000)

Tamaki H.等人：“GPRI 通过 FLOII 调节酿酒酵母中的丝状生长”Biochem。

H.Suzuki, and H.Kumagai.: "γ-Glutamyltranspeptidase : A new member of Ntn hydrolase superfamily."Protein, Nuleic Acid and Enzyme. (in press). (2001)

H.Suzuki 和 H.Kumagai.：“γ-谷氨酰转肽酶：Ntn 水解酶超家族的新成员。”蛋白质、核酸和酶（印刷中）。

Katayama T.: "Cloning and random mutagenesis of the erwinia herbicola tyrR gene for high-level expression of tyrosine phenol-lyase"Appl Environ Microbiol.. 66. 4764-4771 (2000)

Katayama T.：“用于高水平表达酪氨酸酚裂解酶的草欧文氏菌 tyrR 基因的克隆和随机诱变”Appl Environ Microbiol.. 66. 4764-4771 (2000)

Kenji Yamamoto et al.: "Effect of carbohydrate chain on surface net charge and hydrophobicity of glycoproteins."Biosci.Biotech.Biochem.. 62(11). 2171-2176 (1998)

Kenji Yamamoto 等人：“碳水化合物链对糖蛋白表面净电荷和疏水性的影响。”Biosci.Biotech.Biochem.. 62(11)。

熊谷英彦 ら: "ビフィズス菌由来の固定化酵素によるビフィズス因子の合成"ビタミン. 7. 403-405 (1999)

Hidehiko Kumagai 等人：“使用源自双歧杆菌的固定化酶合成双歧因子”维生素。7. 403-405 (1999)