Redesign of the coenzyme specificity in L-lactate dehydrogenase from bacillus stearothermophilus using site-directed mutagenesis and media engineering.
L-lactate dehydrogenase (LDH) from Bacillus stearothermophilus is a redox enzyme which has a strong preference for NADH over NADPH as coenzyme. To exclude NADPH from the coenzyme-binding pocket, LDH contains a conserved aspartate residue at position 52. However, this residue is probably not solely responsible for the NADH specificity. In this report we examine the possibilities of altering the coenzyme specificity of LDH by introducing a range of different point mutations in the coenzyme-binding domain. Furthermore, after choosing the mutant with the highest selectivity for NADPH, we also investigated the possibility of further altering the coenzyme specificity by adding an organic solvent to the reaction mixture. The LDH mutant, I51K:D52S, exhibited a 56-fold increased specificity to NADPH over the wild-type LDH in a reaction mixture containing 15% methanol. Furthermore, the NADPH turnover number of this mutant was increased almost fourfold as compared with wild-type LDH. To explain the altered coenzyme specificity exhibited by the D52SI51K double mutant, molecular dynamics simulations were performed.
Holmberg
N
N
Department of Pure and Applied Biochemistry and Department of Theoretical Chemistry, Centre for Chemistry and Chemical Engineering, Lund University, PO Box 124, S-221 00 Lund, Sweden.
Ryde
U
U
Bülow
L
L
D020816:Amino Acid Motifs / Q000235:genetics; D001665:Binding Sites / Q000235:genetics; D003001:Cloning, Molecular; D003067:Coenzymes / Q000378:metabolism*; D003198:Computer Simulation; D003470:Culture Media; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation / Q000187:drug effects; D001411:Geobacillus stearothermophilus / Q000201:enzymology* / Q000235:genetics; D007770:L-Lactate Dehydrogenase / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D000432:Methanol / Q000494:pharmacology; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009243:NAD / Q000378:metabolism; D009249:NADP / Q000378:metabolism; D015202:Protein Engineering; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D013379:Substrate Specificity
D003067:Coenzymes; D003470:Culture Media; D011994:Recombinant Proteins; D009243:NAD; D009249:NADP; D007770:L-Lactate Dehydrogenase; D000432:Methanol