Evolutional design of a hyperactive cysteine- and methionine-free mutant of Escherichia coli dihydrofolate reductase
Evolutional design of a hyperactive cysteine- and methionine-free mutant of Escherichia coli dihydrofolate reductase
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DOI：
10.1074/jbc.m508823200
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发表时间：
2006-05-12
影响因子：
4.8
通讯作者：
Gekko, K
中科院分区：
文献类型：
--
作者：
Iwakura, M;Maki, K;Gekko, K
我们开发了一种策略，找出适应的酶的变体，我们将其应用于酶，二氢叶酸还原酶（DHFR），在其催化活性方面，使我们成功地获得了几个超活性的半胱氨酸和蛋氨酸自由的变体DHFR中的所有五个甲硫氨酰和两个半胱氨酰残基被替换为其他氨基酸残基。其中，被命名为ANLYF的变体（M1A/M16 N/M20 L/M42 Y/C85 A/M92 F/C152 S）具有比野生型DHFR高约7倍的k（cat）值。对该变体的酶动力学和晶体结构进行了研究，以阐明其活性亢进的机制。稳态和瞬态结合动力学的变体表明，动力学方案的催化循环的ANLYF基本上是相同的野生型，表明的超活性所带来的增加的解离速率常数的四氢叶酸从酶-NADPH-四氢叶酸三元复合物。在1.9-A分辨率下解析并精修至R因子为0.205的变体的晶体结构表明，变体的结构灵活性增加和N-（对氨基苯甲酰基）-L-谷氨酸结合裂缝的大小增加诱导解离常数增加。这与变体的大可压缩性（体积波动）一致。野生型和变异体之间的折叠动力学的比较表明，这两种酶的折叠是彼此相似的，这表明可以实现酶的活性增强而不剧烈改变的折叠机制。
We developed a strategy for finding out the adapted variants of enzymes, and we applied it to an enzyme, dihydrofolate reductase ( DHFR), in terms of its catalytic activity so that we successfully obtained several hyperactive cysteine- and methionine-free variants of DHFR in which all five methionyl and two cysteinyl residues were replaced by other amino acid residues. Among them, a variant ( M1A/M16N/M20L/M42Y/C85A/M92F/C152S), named as ANLYF, has an approximately seven times higher k(cat) value than wild type DHFR. Enzyme kinetics and crystal structures of the variant were investigated for elucidating the mechanism of the hyperactivity. Steady-state and transient binding kinetics of the variant indicated that the kinetic scheme of the catalytic cycle of ANLYF was essentially the same as that of wild type, showing that the hyperactivity was brought about by an increase of the dissociation rate constants of tetrahydrofolate from the enzyme-NADPH-tetrahydrofolate ternary complex. The crystal structure of the variant, solved and refined to an R factor of 0.205 at 1.9-A resolution, indicated that an increased structural flexibility of the variant and an increased size of the N-( p-aminobenzoyl)-L-glutamate binding cleft induced the increase of the dissociation constant. This was consistent with a large compressibility ( volume fluctuation) of the variant. A comparison of folding kinetics between wild type and the variant showed that the folding of these two enzymes was similar to each other, suggesting that the activity enhancement of the enzyme can be attained without drastic changes of the folding mechanism.