Use of Cryo-EM To Uncover Structural Bases of pH Effect and Cofactor Bispecificity of Ketol-Acid Reductoisomerase
Use of Cryo-EM To Uncover Structural Bases of pH Effect and Cofactor Bispecificity of Ketol-Acid Reductoisomerase
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DOI：
10.1021/jacs.9b01354
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发表时间：
2019-04-17
影响因子：
15
通讯作者：
Tsai, Ming-Daw
中科院分区：
文献类型：
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作者：
Chen, Chin-Yu;Chang, Yuan-Chih;Tsai, Ming-Daw
虽然冷冻-EM正在给结构生物学带来革命性的变化，但它对酶学的影响尚未得到充分证明。酮酸还原异构酶(KARI)催化(2S)-乙酰乳酸或(2S)-乙酰基-2-羟基丁酸酯转化为2，3-二羟基-3-烷基丁酸酯。我们发现，古生菌Sulfolobus solfararicus(SSO-KARI)的KARI是一个不寻常的十二聚体，与NADH和NADPH具有双特异性，并且在pH 7.8以上失去活性。SsoKARI：2 mg(2+)在pH 7.5和8.5的条件下均可获得晶体。结果表明，在pH值为8.5时，两种结构中的两种催化镁离子的距离都有所延长。接下来，我们解决了两个SsoKARI络合物的低温EM结构，它们分别含有NADH+抑制剂和NADPH+抑制剂，这表明这种双特异性可以归因于辅因子结合环上的一个独特的天冬酰胺。出乎意料的是，SSO-KARI还与其他KARI酶的不同之处在于缺乏“诱导配合”，体现了结构刚性。因此，冷冻-EM在结构和机械酶学方面是强大的。
While cryo-EM is revolutionizing structural biology, its impact on enzymology is yet to be fully demonstrated. The ketol-acid reductoisomerase (KARI) catalyzes conversion of (2S)-acetolactate or (2S)-aceto-2-hydroxybutyrate to 2,3-dihydroxy-3-alkylbutyrate. We found that KARI from archaea Sulfolobus solfataricus (Sso-KARI) is unusual in being a dodecamer, bispecific to NADH and NADPH, and losing activity above pH 7.8. While crystals were obtainable only at pH 8.5, cryo-EM structures were solved at pH 7.5 and 8.5 for SsoKARI:2Mg(2+). The results showed that the distances of the two catalytic Mg2+ ions are lengthened in both structures at pH 8.5. We next solved cryo-EM structures of two SsoKARI complexes, with NADH+inhibitor and NADPH+inhibitor at pH 7.5, which indicate that the bispecificity can be attributed to a unique asparagine at the cofactor binding loop. Unexpectedly, Sso-KARI also differs from other KARI enzymes in lacking "induced-fit", reflecting structural rigidity. Thus, cryo-EM is powerful for structural and mechanistic enzymology.