Restoration of enzymatic activity in frog lens rho-crystallin

从青蛙晶状体cDNA文库中分离到编码Rho晶体蛋白的克隆(R7Ff)。利用R7FF衍生的表达载体pMR在大肠杆菌(HB101)中表达成熟型Rho-crystallin-II。重组Rho-晶体蛋白的N-末端和C-末端与从青蛙晶状体中纯化的Rho-晶体蛋白-II的N-末端和C-末端完全相同。在前列腺素F合成酶、醛还原酶和醛还原酶等醛酮还原酶中，55-苏氨酸是Rho-cystallin的专一性酶，酪氨酸在所有其他醛酮还原酶中是保守的。因此，我们制备了55-酪氨酸-Rho-晶体蛋白突变体，该突变体具有醛酮还原酶活性。结果表明，55-苏氨酸单点突变为酪氨酸可使Rho-晶体蛋白的酶活性恢复。以9，10-菲醌为底物，在pH 6.5条件下，该突变株在30ﾟC时的比活为0.63U/mg蛋白质，并有葡萄糖还原现象。突变酶的活性与醛糖还原酶的活性相对应，这与糖尿病并发症的形成有关。在Rho-晶体蛋白的分子进化过程中，可能会发生55-酪氨酸到苏氨酸的点突变，以避免醛糖还原酶的活性。

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.