Enhancement of the latent 3-isopropylmalate dehydrogenase activity of promiscuous homoisocitrate dehydrogenase by directed evolution.
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.
Suzuki
Yumewo
Y
Biotechnology Research Center, University of Tokyo, Bunkyo-ku, Japan.
Asada
Kuniko
K
Miyazaki
Junichi
J
Tomita
Takeo
T
Kuzuyama
Tomohisa
T
Nishiyama
Makoto
M
D050539:3-Isopropylmalate Dehydrogenase / Q000737:chemistry / Q000378:metabolism*; D000429:Alcohol Oxidoreductases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D018360:Crystallography, X-Ray; D004789:Enzyme Activation; D004795:Enzyme Stability; D007700:Kinetics; D008958:Models, Molecular; D009154:Mutation; D020836:Protein Structure, Quaternary; D017434:Protein Structure, Tertiary; D040681:Structural Homology, Protein; D013379:Substrate Specificity; D016963:Thermus thermophilus / Q000201:enzymology*
D000429:Alcohol Oxidoreductases; D050539:3-Isopropylmalate Dehydrogenase; C021368:homoisocitrate dehydrogenase