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 alpha-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-succiny1-3,4-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 alpha-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. (C) 2014 Elsevier Inc. All rights reserved.