Restoration of enzymatic activity in frog lens rho-crystallin

蛙晶状体蛋白酶活性的恢复

• 批准号: 06671756
• 负责人: FUJII Yutaka
• 金额: $ 1.41万
• 依托单位: FUKUIMEDICAL SCHOOL
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for General Scientific Research (C)
• 财政年份: 1994
• 资助国家: 日本
• 起止时间: 1994 至 1995
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-06671756/
• 关键词: rho-crystallin; molecular evolution; point mutation; restoration; カエル; アルド・ケト還元酵素; 糖尿病性合併症; ポリオール浸透圧説; プロスタグランジンF合成酵素; ポイントミューテーション

From frog lens cDNA library, the clone (R7FF) encoding rho-crystallin was isolated. Expression vector pMR derived from R7FF was employed for the expression of mature-type rho-crystallin-II in E.coli.(HB101). N- and C-termini of the recombinant rho-crystallin were identical to those of rho-crystallin-II purified from frog lens. Among aldo-keto reductases such as prostaglandin F synthase, aldoes reductase, and aldehyde reductase, 55-threonine is specific for rho-cystallin and tyrosine is conserved among all of other aldo-keto reductases. Therefore, 55-tyr-rho-crystallin mutant was prepared, the mutant showed a aldo-keto reductase activity. It was demonstrated that the restoration of enzymatic activity in rho-crystallin was achieved by the single point mutation of 55-threonine to tyrosine. When 9,10-phenanthrenequinone was used as substrate at pH 6.5, the specific activity of the mutant was determined to be 0.63 units/mg protein at 30ﾟC.Reduction of glucose was also observed in the mutant enzyme. The activity of the mutant enzyme corresponded to that of aldose reductase which contribute to formation of diabetic complications. During the molecular evolution of rho-crystallin, point mutation of 55-tyrosine to threonine may occurred to avoid aldose reductase like activity.

从青蛙晶状体cDNA文库中分离出编码rho-晶状体蛋白的克隆(R7FF)，该表达载体pMR用于在大肠杆菌(HB101)中表达成熟型rho-晶状体蛋白-II。重组rho-晶状体蛋白的C端与醛酮中纯化的rho-晶状体蛋白-II的C端相同。在前列腺素F合酶、醛还原酶和乙醛还原酶等还原酶中，55-苏氨酸对rho-cystallin具有特异性，而酪氨酸在所有其他醛-酮还原酶中是保守的。因此，制备了55-tyr-rho-crystallin突变体。突变体表现出醛酮还原酶活性，表明酶活性恢复。 rho-晶状体蛋白是通过将 55-苏氨酸单点突变为酪氨酸而获得的，当在 pH 6.5 下使用 9,10-菲醌作为底物时，突变体在 30°C 下的比活性为 0.63 单位/mg 蛋白质。在突变酶中也观察到葡萄糖的减少。突变酶的活性与醛糖还原酶的活性相对应，醛糖还原酶有助于形成。在rho-晶状体蛋白的分子进化过程中，可能会发生55-酪氨酸点突变为苏氨酸，以避免醛糖还原酶样活性。

项目成果

YUTAKA FUJII,NOBORU NAKAI,AND HIRONORI THO: "ALDO-KETO REDUCTASE SUPERFAMILY" SEITAINOKAGAKU. 46. 479-480 (1995)

Yutaka Fujii、NOBORU NAKAI 和 HIRONORI THO：“醛酮还原酶超家族”Seitainokagaku。

藤井 豊: "アルド・ケトレダクターゼスーパーファミリー" 生体の科学. 46. 479-480 (1995)

Yutaka Fujii：“醛酮还原酶超家族”生物科学 46. 479-480 (1995)。