title: Structural optimization of SadA, an Fe(II)- and α-ketoglutarate-dependent dioxygenase targeting biocatalytic synthesis of N-succinyl-L-threo-3,4-dimethoxyphenylserine.
abstract: L-threo-3,4-Dihydroxyphenylserine (l-DOPS, Droxidopa) is a psychoactive drug and synthetic amino acid precursor that acts as a prodrug to the neurotransmitters. SadA, a dioxygenase from Burkholderia ambifaria AMMD, is an Fe(II)- and α-ketoglutarate (KG)-dependent enzyme that catalyzes N-substituted branched-chain or aromatic l-amino acids. SadA is able to produce N-succinyl-l-threo-3,4-dimethoxyphenylserine (NSDOPS), which is a precursor of l-DOPS, by catalyzing the hydroxylation of N-succinyl-3,4-dimethoxyphenylalanine (NSDOPA). However, the catalytic activity of SadA toward NSDOPS is much lower than that toward N-succinyl branched-chain l-amino acids. Here, we report an improved biocatalytic synthesis of NSDOPS with SadA. Structure-based protein engineering was applied to improve the α-KG turnover activity for the synthesis of NSDOPS. The G79A, G79A/F261W or G79A/F261R mutant showed a more than 6-fold increase in activity compared to that of the wild-type enzyme. The results provide a new insight into the substrate specificity toward NSDOPA and will be useful for the rational design of SadA mutants as a target of industrial biocatalysts.
authors.affiliation: Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan.
authors.forename: Hui-Min
authors.initials: HM
authors.lastname: Qin
authors.affiliation: Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan.
authors.forename: Takuya
authors.initials: T
authors.lastname: Miyakawa
authors.affiliation: Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan.
authors.forename: Akira
authors.initials: A
authors.lastname: Nakamura
authors.affiliation: Laboratory of Industrial Microbiology, Graduate School of Agriculture, Kyoto University, Kyoto, Japan.
authors.forename: Makoto
authors.initials: M
authors.lastname: Hibi
authors.affiliation: Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Kyoto, Japan.
authors.forename: Jun
authors.initials: J
authors.lastname: Ogawa
authors.affiliation: Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan. Electronic address: amtanok@mail.ecc.u-tokyo.ac.jp.
authors.forename: Masaru
authors.initials: M
authors.lastname: Tanokura
mesh_terms: D055162:Biocatalysis; D005296:Ferrous Compounds / Q000378:metabolism*; D007656:Ketoglutaric Acids / Q000378:metabolism*; D006899:Mixed Function Oxygenases / Q000737:chemistry / Q000378:metabolism*; D062105:Molecular Docking Simulation; D011487:Protein Conformation; D012694:Serine / Q000031:analogs & derivatives* / Q000096:biosynthesis
chemical_list: D005296:Ferrous Compounds; D007656:Ketoglutaric Acids; D012694:Serine; D006899:Mixed Function Oxygenases
keywords: Biocatalyst; DOPA; DOPS; Docking simulation; α-KG turnover activity