Biotechnology Research Center, University of Tokyo, Bunkyo-ku, Japan.
Biochem J. 2010 Nov 1;431(3):401-10. doi: 10.1042/BJ20101246.
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.
HICDH（异柠檬酸脱氢酶）通过α-氨基己二酸参与赖氨酸生物合成，是 IPMDH（3-异丙基苹果酸脱氢酶）的同工酶，后者参与亮氨酸生物合成。TtHICDH（嗜热栖热菌 HICDH）可以识别异柠檬酸和异柠檬酸作为底物，并且还表现出 IPMDH 活性，尽管活性降低了很多。在本研究中，通过 DNA 改组技术进行定向进化，将混杂的 TtHICDH 进化为一种表现出明显 IPMDH 活性的酶。通过 5 次重复的 DNA 改组/筛选，获得了能够使大肠杆菌 C600（leuB⁻）在 2 天内在最小培养基中生长的变体。发现其中一个变体 LR5-1 具有 8 个氨基酸取代，与 TtHICDH 相比，对 3-IPM 的 k(cat)/K(m) 值增加了 65 倍。引入单个回补 H15Y 变化导致 k(cat)/K(m) 值进一步增加，并部分恢复了 LR5-1 降低的耐热性。定点突变揭示了在 LR5-1 中发现的大多数氨基酸取代有效地增加了 IPMDH 活性；在底物结合位点周围的取代有助于提高对 3-IPM 的识别，而远离底物结合位点的其他取代提高了 IPMDH 反应的周转数。LR5-1 的晶体结构在 2.4 Å 分辨率下确定，并揭示了α4 螺旋以适合识别 3-IPM 的疏水性γ部分的方式移位。基于晶体结构，讨论了提高周转率的可能原因。
