title: Engineering substrate specificity of succinic semialdehyde reductase (AKR7A5) for efficient conversion of levulinic acid to 4-hydroxyvaleric acid.
abstract: Engineering enzyme substrate specificity is a promising approach that can expand the applicability of enzymes for the biocatalytic production of industrial chemicals and fuels. In this study, succinic semialdehyde reductase (AKR7A5) was engineered for the conversion of levulinic acid to 4-hydroxyvaleric acid. Levulinic acid is a derivative of cellulosic biomass, and 4-hydroxyvaleric acid is a potential precursor to bio-polymers and fuels. Therefore, the enzymatic conversion of levulinic acid to 4-hydroxyvaleric acid is of special significance in that this conversion could provide a meaningful basis for the bio-production of useful chemicals from cellulosic biomass. In engineering the substrate specificity of the AKR7A5, a rational design approach with the aid of enzyme-substrate interatomic contact analysis was applied. The Met13 residue was selected as a key mutation site, and substitutions of the residue with six hydrophobic amino acids were applied. As a result, four mutants with enhanced catalytic activity toward levulinic acid were obtained, and the most improved mutant, Met13Trp, exhibited a 7.0-fold increase in catalytic efficiency. Additionally, the structural effects of the positive mutations were investigated to analyze the structural basis for the enzyme substrate specificity with the target substrate.
authors.affiliation: The Institute of Molecular Biology and Genetics, Seoul National University, Seoul 151-742, Republic of Korea.
authors.forename: Young Joo
authors.initials: YJ
authors.lastname: Yeon
authors.affiliation: Bio-MAX Institute, Seoul National University, Seoul 151-742, Republic of Korea.
authors.forename: Hyung-Yeon
authors.initials: HY
authors.lastname: Park
authors.affiliation: School of Chemical and Biological Engineering, Seoul National University, Seoul 151-744, Republic of Korea; Bio-MAX Institute, Seoul National University, Seoul 151-742, Republic of Korea. Electronic address: yjyoo@snu.ac.kr.
authors.forename: Young Je
authors.initials: YJ
authors.lastname: Yoo
mesh_terms: D000449:Aldehyde Reductase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000074408:Aldo-Keto Reductases; D000818:Animals; D055162:Biocatalysis; D020134:Catalytic Domain; D007982:Levulinic Acids / Q000378:metabolism*; D008715:Methionine / Q000378:metabolism; D051379:Mice; D062105:Molecular Docking Simulation; D016297:Mutagenesis, Site-Directed / Q000379:methods*; D013379:Substrate Specificity; D014631:Valerates / Q000378:metabolism*
chemical_list: D007982:Levulinic Acids; D014631:Valerates; C469548:4-hydroxyvaleric acid; D008715:Methionine; D000074408:Aldo-Keto Reductases; D000449:Aldehyde Reductase; C000614209:AKR7A5 protein, mouse; C032246:levulinic acid
keywords: 4-Hydroxyvaleric acid; Interatomic contact analysis; Levulinic acid; Rational design; Substrate specificity; Succinic semialdehyde reductase