title: Designs for a broad substrate specificity keto acid dehydrogenase.
abstract: Variations have been made to the structure of the nicotinamide adenine dinucleotide (NAD) dependent L-lactate dehydrogenase from Bacillus stearothermophilus at regions of the enzyme that we believe determine specificity toward different alpha-hydroxy acids (RCHOHCOO-, R = CH3, C2H5, etc.). Two regions of LDH that border the active site (but are not involved in the catalytic reaction) were altered in order to accommodate substrates with hydrophobic side chains larger than that of the naturally preferred substrate, pyruvate (R = CH3). The mutations 102-105GlnLysPro----MetValSer and 236-237AlaAla----GlyGly were made to increase the tolerance for large hydrophobic substrate side chains. The triple and double mutants alone gave little improvement for branched-chain-substituted pyruvates. The five changes together produced a broader substrate specificity alpha-hydroxy acid dehydrogenase, with a 55-fold improved kcat for alpha-ketoisocaproate to a value about 1/14 that of the native enzyme for pyruvate. Rational protein engineering enabled coupled changes in enzyme structure to be obtained with greater probability of success than random mutagenesis.
authors: Molecular Recognition Centre, University of Bristol, U.K.
authors: H M
authors: HM
authors: Wilks
authors: D J
authors: DJ
authors: Halsall
authors: T
authors: T
authors: Atkinson
authors: W N
authors: WN
authors: Chia
authors: A R
authors: AR
authors: Clarke
authors: J J
authors: JJ
authors: Holbrook
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D001411:Geobacillus stearothermophilus / Q000201:enzymology / Q000235:genetics; D007700:Kinetics; D007770:L-Lactate Dehydrogenase / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D011487:Protein Conformation; D015202:Protein Engineering
chemical_list: D001426:Bacterial Proteins; D009838:Oligodeoxyribonucleotides; D007770:L-Lactate Dehydrogenase