Production of an activated form of Bacillus stearothermophilus L-2-hydroxyacid dehydrogenase by directed evolution.
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.
Allen
S J
SJ
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
Holbrook
J J
JJ
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
D005635:Fructosediphosphates; D011994:Recombinant Proteins; D000429:Alcohol Oxidoreductases; C083779:2-hydroxyacid dehydrogenase