Expression, purification, crystallization and preliminary X-ray analysis of a novel N-substituted branched-chain l-amino-acid dioxygenase from Burkholderia ambifaria AMMD

Ferrous ion‐ and α‐ketoglutarate‐dependent dioxygenase from Burkholderia ambifaria AMMD (SadA) catalyzes the C3‐hydroxylation of N‐substituted branched‐chain l‐amino acids, especially N‐succinyl‐l‐leucine, coupled to the conversion of α‐ketoglutarate to succinate and CO2. SadA was expressed in Escherichia coli, purified and crystallized using the sitting‐drop vapour‐diffusion method at 293 K. Crystals of selenomethionine‐substituted SadA were obtained using a reservoir solution containing PEG 3000 as the precipitant at pH 9.5 and diffracted X‐rays to 2.4 Å resolution. The crystal belonged to space group P212121, with unit‐cell parameters a = 49.3, b = 70.9, c = 148.2 Å. The calculated Matthews coefficient (VM = 2.1 Å3 Da−1, 41% solvent content) suggested that the crystal contains two molecules per asymmetric unit.