Expression, purification, crystallization and preliminary X-ray diffraction analysis of dihydrodipicolinate synthase fromBacillus anthracisin the presence of pyruvate

Abstract

Dihydrodipicolinate synthase (DHDPS) catalyses the first committed step in the lysine-biosynthesis pathway in bacteria, plants and some fungi. In this study, the expression of DHDPS from Bacillus anthracis (Ba-DHDPS) and the purification of the recombinant enzyme in the absence and presence of the substrate pyruvate are described. It is shown that DHDPS from B. anthracis purified in the presence of pyruvate yields greater amounts of recombinant enzyme with more than 20-fold greater specific activity compared with the enzyme purified in the absence of substrate. It was therefore sought to crystallize Ba-DHDPS in the presence of the substrate. Pyruvate was soaked into crystals of Ba-DHDPS prepared in 0.2 M sodium fluoride, 20%(w/v) PEG 3350 and 0.1 M bis-tris propane pH 8.0. Preliminary X-ray diffraction data of the recombinant enzyme soaked with pyruvate at a resolution of 2.15 Å are presented. The pending crystal structure of the pyruvate-bound form of Ba-DHDPS will provide insight into the function and stability of this essential bacterial enzyme.

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“…Cloning, Expression, and Purification of Ba-DHDPS-The dapA gene encoding DHDPS from B. anthracis (Sterne strain) was amplified by PCR and cloned into the pET11a expression vector as described elsewhere (22). Briefly, recombinant protein was produced in the host strain E. coli BL21-DE3 as follows.…”

“…Cells were harvested 3 h post-induction and resuspended in 20 mM Tris-HCl, pH 8.0, before lysis by sonication. Ba-DHDPS was subsequently isolated by anion-exchange and hydrophobic interaction liquid chromatography as described elsewhere (22).…”

“…For x-ray data collection, crystals were transferred to reservoir solution containing 20% (v/v) glycerol with 20 mM pyruvate and directly flash frozen in liquid nitrogen. Intensity data were collected at the Australian synchrotron using the MX1 beamline as described before (22).…”

“…We recently reported that the raw enzyme activity and yield of recombinant Ba-DHDPS are significantly increased when the enzyme is purified in the presence of its substrate, pyruvate (22). To examine the effect of pyruvate on the stability of Ba-DHDPS in aqueous solution, thermal denaturation experiments monitored by CD spectroscopy were conducted in the presence and absence of the substrates pyruvate or ASA over the temperature range of 20 -90°C.…”

“…Recent work in our laboratory has shown that the purification of recombinant DHDPS from B. anthracis (Ba-DHDPS) in the presence of its substrate pyruvate increases the yield and specific activity of the final product (22). Therefore, the aim of this study was to thoroughly characterize the effect of pyruvate on the solution properties and structure of Ba-DHDPS.…”