title: Production of an activated form of Bacillus stearothermophilus L-2-hydroxyacid dehydrogenase by directed evolution.
abstract: Bacillus stearothermophillus lactate dehydrogenase (bsLDH) is activated in the presence of fructose 1,6 bisphosphate (FBP). The activator is expensive and representative of the sort of co-factor complications that are undesirable in industrial processes. Three rounds of random mutagenesis and screening produced a mutant (6A) which is almost fully activated in the absence of FBP. Wild-type bsLDH has a K(pyr)(M) of 5 mM in the absence of FBP but when activated (+FBP) the K(pyr)(M) drops to 0.05 mM. The mutant 6A has a K(pyr)(M) of 0.07 mM in the absence of FBP. 6A has three amino acid substitutions-R118C, Q203L and N307S-resulting in a 70-fold activation, none of the mutations are near the active site. The activation of wild type bsLDH is due to an FBP induced tetramerization of dimeric bsLDH bringing about a structural rearrangement of key active site residues. The most likely explanation for the activation of 6A is derived from the position of Q203L, which is at the dimer-dimer interface. The suggestion is that the hydrophilic to hydrophobic change has altered the dimer-tetramer equilibrium position towards that of the tetramer. What is significant is the activation of bsLDH by a subtle long range event produced by the 'blind' directed evolution approach.
authors.affiliation: Molecular Recognition Centre, Department of Biochemistry, School of Medical Sciences, University of Bristol, University Walk,Bristol BS8 1TD, UK. S.J.Allen@bristol.ac.uk
authors.forename: S J
authors.initials: SJ
authors.lastname: Allen
authors.forename: J J
authors.initials: JJ
authors.lastname: Holbrook
mesh_terms: D000429:Alcohol Oxidoreductases / Q000235:genetics* / Q000378:metabolism*; D019943:Amino Acid Substitution; D004789:Enzyme Activation; D005635:Fructosediphosphates; D001411:Geobacillus stearothermophilus / Q000201:enzymology*; D007700:Kinetics; D016296:Mutagenesis; D009154:Mutation; D015202:Protein Engineering / Q000379:methods*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012641:Selection, Genetic; D020539:Sequence Analysis, Protein; D013379:Substrate Specificity
chemical_list: D005635:Fructosediphosphates; D011994:Recombinant Proteins; D000429:Alcohol Oxidoreductases; C083779:2-hydroxyacid dehydrogenase