title: Uncovering rare NADH-preferring ketol-acid reductoisomerases.
abstract: All members of the ketol-acid reductoisomerase (KARI) enzyme family characterized to date have been shown to prefer the nicotinamide adenine dinucleotide phosphate hydride (NADPH) cofactor to nicotinamide adenine dinucleotide hydride (NADH). However, KARIs with the reversed cofactor preference are desirable for industrial applications, including anaerobic fermentation to produce branched-chain amino acids. By applying insights gained from structural and engineering studies of this enzyme family to a comprehensive multiple sequence alignment of KARIs, we identified putative NADH-utilizing KARIs and characterized eight whose catalytic efficiencies using NADH were equal to or greater than NADPH. These are the first naturally NADH-preferring KARIs reported and demonstrate that this property has evolved independently multiple times, using strategies unlike those used previously in the laboratory to engineer a KARI cofactor switch.
authors.affiliation: California Institute of Technology, Division of Chemistry and Chemical Engineering, 1200 E California Blvd, MC 210-41, Pasadena, CA 91125, USA. Electronic address: sabine@cheme.caltech.edu.
authors.forename: S
authors.initials: S
authors.lastname: Brinkmann-Chen
authors.affiliation: California Institute of Technology, Division of Chemistry and Chemical Engineering, 1200 E California Blvd, MC 210-41, Pasadena, CA 91125, USA. Electronic address: jcahn@caltech.edu.
authors.forename: J K B
authors.initials: JKB
authors.lastname: Cahn
authors.affiliation: California Institute of Technology, Division of Chemistry and Chemical Engineering, 1200 E California Blvd, MC 210-41, Pasadena, CA 91125, USA. Electronic address: frances@cheme.caltech.edu.
authors.forename: F H
authors.initials: FH
authors.lastname: Arnold
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites; D004789:Enzyme Activation; D050540:Ketol-Acid Reductoisomerase / Q000737:chemistry*; D008969:Molecular Sequence Data; D009243:NAD / Q000737:chemistry*; D011485:Protein Binding; D016415:Sequence Alignment / Q000379:methods*; D020539:Sequence Analysis, Protein / Q000379:methods*; D013379:Substrate Specificity
chemical_list: D009243:NAD; D050540:Ketol-Acid Reductoisomerase
keywords: Cofactor specificity; Ketol acid reductoisomerase; NADH; NADPH