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 kcat/Km value for 3-IPM, compared with TtHICDH. Introduction of a single back-replacement H15Y change caused a further increase in the kcat/Km 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.
Enhancement of the latent 3-isopropylmalate dehydrogenase activity of promiscuous homoisocitrate dehydrogenase by directed evolution
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Yumewo Suzuki, Kuniko Asada, Junichi Miyazaki, Takeo Tomita, Tomohisa Kuzuyama, Makoto Nishiyama; Enhancement of the latent 3-isopropylmalate dehydrogenase activity of promiscuous homoisocitrate dehydrogenase by directed evolution. Biochem J 1 November 2010; 431 (3): 401–412. doi: https://doi.org/10.1042/BJ20101246
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