title: Substitutions at the cofactor phosphate-binding site of a clostridial alcohol dehydrogenase lead to unexpected changes in substrate specificity.
abstract: Changing the cofactor specificity of an enzyme from nicotinamide adenine dinucleotide 2'-phosphate (NADPH) to the more abundant NADH is a common strategy for increasing overall enzyme efficiency in microbial metabolic engineering. The aim of this study was to switch the cofactor specificity of the primary-secondary alcohol dehydrogenase from Clostridium autoethanogenum, a bacterium with considerable promise for the bio-manufacturing of fuels and other petrochemicals, from strictly NADPH-dependent to NADH-dependent. We used insights from a homology model to build a site-saturation library focussed on residue S199, the position deemed most likely to disrupt binding of the 2'-phosphate of NADPH. Although the CaADH(S199X) library did not yield any NADH-dependent enzymes, it did reveal that substitutions at the cofactor phosphate-binding site can cause unanticipated changes in the substrate specificity of the enzyme. Using consensus-guided site-directed mutagenesis, we were able to create an enzyme that was stringently NADH-dependent, albeit with a concomitant reduction in activity. This study highlights the role that distal residues play in substrate specificity and the complexity of enzyme-cofactor interactions.
authors.affiliation: Department of Biochemistry, University of Otago, Dunedin 9010, New Zealand.
authors.forename: Danielle J
authors.initials: DJ
authors.lastname: Maddock
authors.affiliation: Department of Biochemistry, University of Otago, Dunedin 9010, New Zealand.
authors.forename: Wayne M
authors.initials: WM
authors.lastname: Patrick
authors.affiliation: Department of Biochemistry, University of Otago, Dunedin 9010, New Zealand monica.gerth@otago.ac.nz.
authors.forename: Monica L
authors.initials: ML
authors.lastname: Gerth
mesh_terms: D000426:Alcohol Dehydrogenase / Q000737:chemistry* / Q000235:genetics; D019943:Amino Acid Substitution; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics; D001665:Binding Sites; D003013:Clostridium / Q000201:enzymology*; D013379:Substrate Specificity / Q000235:genetics
chemical_list: D001426:Bacterial Proteins; D000426:Alcohol Dehydrogenase
keywords: Clostridium autoethanogenum; alcohol dehydrogenase; cofactor specificity; enzyme engineering; mutagenesis