3AH3

Crystal structure of LR5-1, 3-isopropylmalate dehydrogenase created by directed evolution

Summary for 3AH3

• Entry DOI: 10.2210/pdb3ah3/pdb
• Related: 1X0L
• Descriptor: Homoisocitrate dehydrogenase, SULFATE ION, 1,2-ETHANEDIOL, ... (4 entities in total)
• Functional Keywords: 3-isopropylmalate dehydrogenase, homoisocitrate dehydrogenase, directed evolution, oxidoreductase
• Biological source: Thermus thermophilus
• Total number of polymer chains: 4
• Total formula weight: 144516.33

Authors

Tomita, T.,Suzuki, Y.,Kuzuyama, T.,Nishiyama, M. (deposition date: 2010-04-13, release date: 2011-02-02, Last modification date: 2023-11-01)

Primary citation

Suzuki, Y.,Asada, K.,Miyazaki, J.,Tomita, T.,Kuzuyama, T.,Nishiyama, M.
Enhancement of the latent 3-isopropylmalate dehydrogenase activity of promiscuous homoisocitrate dehydrogenase by directed evolution
Biochem.J., 431:401-410, 2010

PubMed Abstract: 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.

PubMed: 20735360
DOI: 10.1042/BJ20101246

Experimental method

X-RAY DIFFRACTION (2.4 Å)