Cloning to crystallization of dihydrodipicolinate synthase from the intracellular pathogenLegionella pneumophila

Abstract

Dihydrodipicolinate synthase (DHDPS) catalyses the rate-limiting step in the biosynthesis of meso-diaminopimelate and lysine. Here, the cloning, expression, purification and crystallization of DHDPS from the intracellular pathogen Legionella pneumophila are described. Crystals grown in the presence of highmolecular-weight PEG precipitant and magnesium chloride were found to diffract beyond 1.65 Å resolution. The crystal lattice belonged to the hexagonal space group P6 1 22, with unit-cell parameters a = b = 89.31, c = 290.18 Å , and contained two molecules in the asymmetric unit. The crystal structure was determined by molecular replacement using a single chain of Pseudomonas aeruginosa DHDPS as the search model.

Structural Determinants Defining the Allosteric Inhibition of an Essential Antibiotic Target

“…Cloning, Expression, and Purification of DHDPS Genomic DNA from L. pneumophila (strain Alcoy) and a clinical isolate of S. pneumoniae (serotype 3) were used in this study. The procedures for amplifying and cloning the dapA gene (encoding DHDPS) from L. pneumophila and S. pneumoniae and for the overexpression and purification of recombinant DHDPS were performed as described previously (Atkinson et al, 2011;Burgess et al, 2008b;Dogovski et al, 2013;Sibarani et al, 2010;Siddiqui et al, 2013). Briefly, dapA encoding DHDPS was PCR-amplified from genomic DNA and cloned into pET11a, pET28a, or pRSET A expression vector (Table 1).…”

“…Crystallization, Structure Determination, and Refinement X-Ray diffraction experiments were carried out at the Australian Synchrotron on the MX1 beamline (Cowieson et al, 2015) using Blu-Ice (McPhillips et al, 2002). LpDHDPS was crystallized using the hanging-drop vapor diffusion method as previously described (Siddiqui et al, 2013). Crystals were soaked in reservoir solution containing 20% (v/v) 2-methyl-2,4-pentanediol (MPD) prior to data collection.…”

Molecular evolution of an oligomeric biocatalyst functioning in lysine biosynthesis

“…In contrast to other enzymes in the DAP pathway, the expression of dapA in E. coli was not found to be regulated by cellular free lysine levels or any other stimuli (Butour et al 1974). In the last three decades, the dapA gene has been cloned and sequenced from a variety of other bacterial (Atkinson et al 2011(Atkinson et al , 2012aChen et al 1993;Cremer et al 1988;Devenish et al 2009;Dommaraju et al 2010;Evans et al 2011;García-Rodríguez et al 2000;Girish et al 2008;Gunji et al 2004;Kaur et al 2011;Pisabarro et al 1993;Siddiqui et al 2013;Skovpen and Palmer 2013;Wolterink-van Loo et al 2008;Wubben et al 2010) and plant species (Atkinson et al 2011(Atkinson et al , 2014Frisch et al 1991;Ghislain et al 1995;Kaneko et al 1990;Silk et al 1994;Vauterin and Jacobs 1994). Typically, bacteria have a single dapA gene that is 800-900 bp, whereas plants have two annotated dapA genes consisting of~1000 bp each.…”