Restoration of enzymatic activity in frog lens rho-crystallin

从蛙透镜cDNA文库中克隆了一个编码rho-crystallin的克隆（R7 FF）。利用来源于R7 FF的表达载体pMR在大肠杆菌中表达成熟型rh-crystallin-II。（HB101）。重组rh-crystallin的N-和C-末端与从青蛙透镜中纯化的rh-crystallin-II的相同。在醛-酮还原酶如前列腺素F合酶、醛还原酶和醛还原酶中，55-苏氨酸对rho-胱抑素具有特异性，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，该突变体表现出醛酮还原酶活性。结果表明，55-苏氨酸突变为酪氨酸后，可使rh-crystallin酶活性恢复。当9，10-菲醌在pH 6.5下用作底物时，突变体的比活性在30 ° C下被确定为0.63单位/mg蛋白质。在突变体酶中还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，这有助于糖尿病并发症的形成。在rho-crystallin的分子进化过程中，可能发生了55-酪氨酸到苏氨酸的点突变，以避免类似醛糖还原酶的活性。

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.