Structural optimization of SadA, an Fe(II)- and α-ketoglutarate-dependent dioxygenase targeting biocatalytic synthesis of N-succinyl-l-threo-3,4-dimethoxyphenylserine.

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. [ABSTRACT FROM AUTHOR]