Crystal Structure of a Novel N-Substituted L-Amino Acid Dioxygenase from Burkholderia ambifaria AMMD

Abstract

A novel dioxygenase from Burkholderia ambifaria AMMD (SadA) stereoselectively catalyzes the C3-hydroxylation of N-substituted branched-chain or aromatic L-amino acids, especially N-succinyl-L-leucine, coupled with the conversion of α-ketoglutarate to succinate and CO2. To elucidate the structural basis of the substrate specificity and stereoselective hydroxylation, we determined the crystal structures of the SadA.Zn(II) and SadA.Zn(II).α-KG complexes at 1.77 Å and 1.98 Å resolutions, respectively. SadA adopted a double-stranded β-helix fold at the core of the structure. In addition, an HXD/EXnH motif in the active site coordinated a Zn(II) as a substitute for Fe(II). The α-KG molecule also coordinated Zn(II) in a bidentate manner via its 1-carboxylate and 2-oxo groups. Based on the SadA.Zn(II).α-KG structure and mutation analyses, we constructed substrate-binding models with N-succinyl-L-leucine and N-succinyl-L-phenylalanine, which provided new insight into the substrate specificity. The results will be useful for the rational design of SadA variants aimed at the recognition of various N-succinyl L-amino acids.

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

“…The expression and purification of SadA were performed according to previous reports [14,15]. In brief, Escherichia coli Rosetta (DE3) cells (Novagen, Madison, WI) were transformed with the pQE80 plasmid harboring SadA sequence (pQE80-SadA) and were grown in lysogeny broth (LB) medium at 37°C.…”

“…The initial model of SadA.Zn(II).a-KG.NSDOPA was constructed using the Molecule Builder of the molecular operating environment (MOE; Chemical Computing Group, Montreal, Canada) based on the model of binding between SadA.Zn(II).a-KG and NSPhe [15]. Fe(II) was replaced by Zn(II) in the model based on the crystal structure reported previously [15].…”

“…Fe(II) was replaced by Zn(II) in the model based on the crystal structure reported previously [15]. For the binding model of the G79A, G79A/F261W and G79A/F261R mutants, individual residues identified by the MOE site finder search were substituted into the initial model.…”

Structural analysis of enzymes used for bioindustry and bioremediation

“…[37][38][39] The SadA structure provides the mechanistic basis for the stereoselective reaction of NSLeu to make the expansion of substrate specificity possible. 40,41) SadA forms a dimeric structure, and each protomer adopts a double-stranded β-helix (DSBH) fold at the core of the structure, along with six α-helices (Fig. 4(A)).…”

“…40) Their importance in the substrate specificity is supported by the result that the F261A mutant exhibits increased activity toward N-succinyl-L-3,4-dimethoxyphenylalanine (NSDOPA), 41) which is converted to N-succinyl-L-threo-3,4-dihydroxyphenylserine, a precursor of L-DOPS, and possesses a bulkier sidechain than NSLeu. The results also show that the structural information is useful for the improvement of substrate specificity, which is a major target of the rational design of useful enzymes based on protein engineering.…”