title: Attempting to remove the substrate inhibition of L-lactate dehydrogenase from Bacillus stearothermophilus by site-directed mutagenesis.
abstract: L-lactate dehydrogenase (LDH) catalyzes the interconversion of an oxoacid (pyruvate) and hydroxy-acid (lactate) using the NADH/NAD+ pair as a redox cofactor. The enzyme has a commercial significance, as it can be used to produce chiral building blocks for the synthesis of key pharmaceuticals and agrochemicals. However, the substrate inhibition which is due to an abortive NAD+-pyruvate complex reducing the steady state concentration of functional LDH limits its use in industry. This substrate inhibition can be overcome by weaking the binding of NAD+. The conserved aspartic acid residue at position 38 was replaced by the longer basic arginine side chain (D38R) using PCR based overlap extension mutagenesis technique in the hope of weakening NAD+-binding. The mutant gene was overexpressed in the Escherichia coli high-expression vector pKK223-3 in JM105 cells; then, the mutant protein was produced. Comparing the effect of substrate inhibition in the arginine-38 mutant with wild-type, substrate inhibition is decreased threefold.
authors.affiliation: Istanbul Teknik Universitesi, Fen-Edebiyat Fakültesi, Moleküler Biyoloji ve Genetik Bölümü, Istanbul, Turkey.
authors.forename: Bariş
authors.initials: B
authors.lastname: Binay
authors.forename: Nevin Gül
authors.initials: NG
authors.lastname: Karagüler
mesh_terms: D001120:Arginine; D001671:Biochemistry / Q000379:methods; D001709:Biotechnology / Q000379:methods*; D002384:Catalysis; D002478:Cells, Cultured; D004926:Escherichia coli / Q000378:metabolism; D001411:Geobacillus stearothermophilus / Q000201:enzymology*; D007700:Kinetics; D007770:L-Lactate Dehydrogenase / Q000037:antagonists & inhibitors* / Q000737:chemistry*; D008954:Models, Biological; D008956:Models, Chemical; D016296:Mutagenesis; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D009243:NAD / Q000737:chemistry; D015202:Protein Engineering / Q000379:methods*; D013379:Substrate Specificity
chemical_list: D009243:NAD; D001120:Arginine; D007770:L-Lactate Dehydrogenase