Open AccessExpression, purification, crystallization and preliminary X‐ray analysis of a novel N‐substituted branched‐chain l ‐amino‐acid dioxygenase from Burkholderia ambifaria AMMD
Author(s) -
Qin HuiMin,
Miyakawa Takuya,
Nakamura Akira,
Xue YouLin,
Kawashima Takashi,
Kasahara Takuya,
Hibi Makoto,
Ogawa Jun,
Tanokura Masaru
Publication year - 2012
Publication title -
acta crystallographica section f
Language(s) - English
Resource type - Journals
ISSN - 1744-3091
DOI - 10.1107/s1744309112031508
Subject(s) - dioxygenase , crystallization , hydroxylation , chemistry , escherichia coli , amino acid , molecule , crystal structure , solvent , crystal (programming language) , stereochemistry , crystallography , biochemistry , enzyme , organic chemistry , programming language , gene , computer science
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 CO 2 . 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 P 2 1 2 1 2 1 , with unit‐cell parameters a = 49.3, b = 70.9, c = 148.2 Å. The calculated Matthews coefficient ( V M = 2.1 Å 3 Da −1 , 41% solvent content) suggested that the crystal contains two molecules per asymmetric unit.