Open AccessOverexpression, crystallization and preliminary X‐ray crystallographic analysis of shikimate dehydrogenase from Archaeoglobus fulgidus
Author(s) -
Lee Hyung Ho
Publication year - 2011
Publication title -
acta crystallographica section f
Language(s) - English
Resource type - Journals
ISSN - 1744-3091
DOI - 10.1107/s1744309111041194
Subject(s) - shikimate pathway , chemistry , crystallography , ammonium sulfate , crystallization , escherichia coli , stereochemistry , biochemistry , enzyme , biosynthesis , gene , organic chemistry
Shikimate dehydrogenase (SDH), which catalyses the NADPH‐dependent reduction of 3‐dehydroshikimate to shikimate in the shikimate pathway, is an attractive target for the development of herbicides and antimicrobial agents. Previous structural studies have shown that SDH exists in two conformations, an open and a closed form, and it is believed that the conformational state is crucial to understanding its catalytic mechanism. In order to facilitate further structural comparisons among SDHs, including the conformational state, structural analysis of an SDH from Archaeoglobus fulgidus encoded by the Af2327 gene has been initiated. SeMet‐labelled SDH from A. fulgidus was overexpressed in Escherichia coli and crystallized at 296 K using ammonium sulfate as a precipitant in order to use the MAD method for structure determination. Crystals of A. fulgidus SDH grown in the presence of NADP + diffracted to 2.8 Å resolution and belonged to the trigonal space group P 3 2 21 (or P 3 2 21), with unit‐cell parameters a = 111.3, b = 111.3, c = 76.2 Å. Three diffraction data sets were collected. The asymmetric unit contains two monomers, with a corresponding V M of 2.34 Å 3 Da −1 and a solvent content of 47% by volume.