title: General approach to reversing ketol-acid reductoisomerase cofactor dependence from NADPH to NADH.
abstract: To date, efforts to switch the cofactor specificity of oxidoreductases from nicotinamide adenine dinucleotide phosphate (NADPH) to nicotinamide adenine dinucleotide (NADH) have been made on a case-by-case basis with varying degrees of success. Here we present a straightforward recipe for altering the cofactor specificity of a class of NADPH-dependent oxidoreductases, the ketol-acid reductoisomerases (KARIs). Combining previous results for an engineered NADH-dependent variant of Escherichia coli KARI with available KARI crystal structures and a comprehensive KARI-sequence alignment, we identified key cofactor specificity determinants and used this information to construct five KARIs with reversed cofactor preference. Additional directed evolution generated two enzymes having NADH-dependent catalytic efficiencies that are greater than the wild-type enzymes with NADPH. High-resolution structures of a wild-type/variant pair reveal the molecular basis of the cofactor switch.
authors.affiliation: California Institute of Technology, Pasadena, CA 91125, USA.
authors.forename: Sabine
authors.initials: S
authors.lastname: Brinkmann-Chen
authors.forename: Tilman
authors.initials: T
authors.lastname: Flock
authors.forename: Jackson K B
authors.initials: JK
authors.lastname: Cahn
authors.forename: Christopher D
authors.initials: CD
authors.lastname: Snow
authors.forename: Eric M
authors.initials: EM
authors.lastname: Brustad
authors.forename: John A
authors.initials: JA
authors.lastname: McIntosh
authors.forename: Peter
authors.initials: P
authors.lastname: Meinhold
authors.forename: Liang
authors.initials: L
authors.lastname: Zhang
authors.forename: Frances H
authors.initials: FH
authors.lastname: Arnold
mesh_terms: D000595:Amino Acid Sequence; D019020:Directed Molecular Evolution; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D050540:Ketol-Acid Reductoisomerase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009243:NAD / Q000378:metabolism; D009249:NADP / Q000378:metabolism; D017433:Protein Structure, Secondary; D017386:Sequence Homology, Amino Acid
chemical_list: D029968:Escherichia coli Proteins; D009243:NAD; D009249:NADP; D050540:Ketol-Acid Reductoisomerase
keywords: branched-chain amino acid pathway; cofactor imbalance