Enhancement of the latent 3-isopropylmalate dehydrogenase activity of promiscuous homoisocitrate dehydrogenase by directed evolution.
Enhancement of the latent 3-isopropylmalate dehydrogenase activity of promiscuous homoisocitrate dehydrogenase by directed evolution.
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DOI：
10.1042/bj20101246
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发表时间：
2010-11
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通讯作者：
Yumewo Suzuki;Kuniko Asada;J. Miyazaki;T. Tomita;T. Kuzuyama;M. Nishiyama
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作者：
Yumewo Suzuki;Kuniko Asada;J. Miyazaki;T. Tomita;T. Kuzuyama;M. Nishiyama
高异柠檬酸脱氢酶（homoisocitrate dehydrogenase，HIDDH）是亮氨酸生物合成中的3-异丙基苹果酸脱氢酶（IPMDH）的一种，通过α-氨基己二酸参与赖氨酸的生物合成。TtMDH（Thermus thermophilus TtMDH）可以识别异柠檬酸盐以及高异柠檬酸盐作为底物，并且还显示IPMDH活性，尽管速率显著降低。在本研究中，混杂的TtdR DH进化成一种酶，显示不同的IPMDH活性的定向进化使用DNA改组技术。通过5次重复的DNA改组/筛选，获得了允许大肠杆菌C600（leuB）在基本培养基上在2天内生长的变体。其中一个具有8个氨基酸替换的变体LR 5 -1被发现具有与TtdR DH相比增加65倍的3-IPM的k（cat）/K（m）值。引入单一回代H15 Y变化导致k（cat）/K（m）值进一步增加，并部分恢复LR 5 -1降低的耐热性。定点突变表明，在LR 5 -1中发现的大多数氨基酸替换有效地增加了IPMDH活性;底物结合位点周围的替换有助于提高对3-IPM的识别，而远离底物结合位点的其他替换增强了IPMDH反应的营业额。在2.4 nm分辨率下测定了LR 5 -1的晶体结构，并显示螺旋α4以适合于识别3-IPM的疏水性γ-部分的方式被置换。在晶体结构的基础上，讨论了翻转数提高的可能原因。
HICDH (homoisocitrate dehydrogenase), which is involved in lysine biosynthesis through α-aminoadipate, is a paralogue of IPMDH [3-IPM (3-isopropylmalate) dehydrogenase], which is involved in leucine biosynthesis. TtHICDH (Thermus thermophilus HICDH) can recognize isocitrate, as well as homoisocitrate, as the substrate, and also shows IPMDH activity, although at a considerably decreased rate. In the present study, the promiscuous TtHICDH was evolved into an enzyme showing distinct IPMDH activity by directed evolution using a DNA-shuffling technique. Through five repeats of DNA shuffling/screening, variants that allowed Escherichia coli C600 (leuB⁻) to grow on a minimal medium in 2 days were obtained. One of the variants LR5-1, with eight amino acid replacements, was found to possess a 65-fold increased k(cat)/K(m) value for 3-IPM, compared with TtHICDH. Introduction of a single back-replacement H15Y change caused a further increase in the k(cat)/K(m) value and a partial recovery of the decreased thermotolerance of LR5-1. Site-directed mutagenesis revealed that most of the amino acid replacements found in LR5-1 effectively increased IPMDH activity; replacements around the substrate-binding site contributed to the improved recognition for 3-IPM, and other replacements at sites away from the substrate-binding site enhanced the turnover number for the IPMDH reaction. The crystal structure of LR5-1 was determined at 2.4 Å resolution and revealed that helix α4 was displaced in a manner suitable for recognition of the hydrophobic γ-moiety of 3-IPM. On the basis of the crystal structure, possible reasons for enhancement of the turnover number are discussed.