| |
[摘要]：HICDH (homoisocitrate dehydrogenase) which is involved in lysine biosynthesis through alpha-ammoadipate is a paralogue of IPMDH [3-IPM (3-isopropylmalate) dehydrogenase], which is involved in leucine biosynthesis TtHICDH (Thermus thermoplulus 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 colt C600 (leu(-)) 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 angstrom resolution and revealed that helix alpha 4 was displaced in a manner suitable for recognition of the hydrophobic gamma--moiety of 3-IPM On the basis of the crystal structure, possible reasons for enhancement of the turnover number are discussed |
|
