Restoration of enzymatic activity in frog lens rho-crystallin

从蛙晶状体cDNA文库中分离到编码R7FF的克隆。利用R7FF衍生的pMR表达载体在大肠杆菌（HB101）中表达成熟型rho-crystallin-II。重组rho-crystallin- ii的N端和c端与从青蛙晶状体中纯化的rho-crystallin- ii相同。在前列腺素F合成酶、醛酮还原酶和醛酮还原酶等醛酮还原酶中，55-苏氨酸对红晶体蛋白是特异性的，而酪氨酸在所有其他醛酮还原酶中都是保守的。因此，我们制备了55- tyrr -rho-crystallin突变体，该突变体具有醛酮还原酶活性。结果表明，通过将55-苏氨酸单点突变为酪氨酸，可以恢复红晶蛋白的酶活性。当pH为6.5时，以9,10-菲醌为底物，在30‰的温度下，突变体的比活性为0.63单位/毫克蛋白。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一致，醛糖还原酶有助于糖尿病并发症的形成。在红结晶蛋白的分子进化过程中，可能发生55-酪氨酸向苏氨酸的点突变，以避免醛糖还原酶样的活性。

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.