Cloning, expression, purification, crystallization and preliminary crystallographic studies of BceC, a UDP-glucose dehydrogenase fromBurkholderia cepaciaIST408

Abstract

Bacteria of the Burkholderia cepacia complex (Bcc) have emerged as important opportunistic pathogens, establishing lung infections in immunocompromised or cystic fibrosis patients. Bcc uses polysaccharide‐biofilm production in order to evade the host immune response. The biofilm precursor UDP‐glucuronic acid is produced by a twofold NAD+‐dependent oxidation of UDP‐glucose. In B. cepacia IST408 this enzymatic reaction is performed by the UDP‐glucose dehydrogenase BceC, a 470‐residue enzyme, the production and crystallization of which are described here. The crystals belonged to the orthorhombic space group P212121 and contained four molecules in the asymmetric unit. Their crystallographic analysis at 2.09 Å resolution and a molecular‐replacement study are reported.

Structure of Burkholderia cepacia UDP-Glucose Dehydrogenase (UGD) BceC and Role of Tyr10 in Final Hydrolysis of UGD Thioester Intermediate

“…BceC wild-type and Y10 mutants were expressed and purified as previously described (47). Briefly, the cells harboring the recombinant constructs of pBceC, pBceC_y10f, pBceC_y10s, and pBceC_y10k were grown at 37°C to an A 600 of approximately 0.6 in LB medium supplemented with 100 mg ml Ϫ1 ampicillin and induced at 28°C for 6 h by addition of isopropyl-␤-D-thiogalactopyranoside (IPTG) to a final concentration of 0.3 mM.…”

“…Point mutants of the BceC protein were generated from the wild-type pBceC construct using a protocol based on a QuickChange site-directed mutagenesis kit (Stratagene, La Jolla, CA) (47). Based on the nucleotide sequence of the bceC gene (GenBank accession number GQ451909), the oligonucleotide pairs Y10S_up (5Ј-ATC ATC GGC AGC GGT TCC GTA GGT CTT GTC ACC-3Ј) and Y10S_rev (5Ј-GGT GAC AAG ACC TAC GGA ACC GCT GCC GAT GAT-3Ј), Y10K_up (5Ј-CT ATC ATC GGC AGC GGT AAG GTA GGT CTT GTC ACC GG-3Ј) and Y10K_rev (5Ј-CC GGT GAC AAG ACC TAC CTT ACC GCT GCC GAT GAT AG-3Ј), and Y10F_up (5Ј-CT ATC ATC GGC AGC GGT TTC GTA GGT CTT GTC AC-3Ј) and Y10F_rev (5Ј-GT GAC AAG ACC TAC GAA ACC GCT GCC GAT GAT AG-3Ј) were designed for mutants Y10S, Y10K, and Y10F, respectively.…”

“…23%. However, once a preliminary three-dimensional (3D) model of UGD from Sphingomonas elodea ATCC 31461 was obtained in our laboratory (46), with a sequence identity of 42% against BceC, the molecular replacement solution was readily obtained (47). Using the initial diffraction data set to 2.09-Å resolution, the phases of the native BceC molecular replacement solution were improved (47) with the program RESOLVE using its automatically implemented 2-fold noncrystallographic symmetry (NCS) averaging and "prime and switch" procedures (54).…”

“…Native BceC crystallization was described elsewhere (47). In spite of extensive crystallization screens, no crystals of the Y10F mutant were ever obtained, but suitable crystals of the Y10S and Y10K mutants were readily produced by optimization of the native BceC sitting-drop vapor diffusion crystallization conditions (47).…”

“…Native BceC crystallization was described elsewhere (47). In spite of extensive crystallization screens, no crystals of the Y10F mutant were ever obtained, but suitable crystals of the Y10S and Y10K mutants were readily produced by optimization of the native BceC sitting-drop vapor diffusion crystallization conditions (47). The best Y10S and Y10K crystals were obtained by equilibration of 1 l of mutant protein solution and 1 l of a precipitant solution of 100 mM sodium acetate buffer, pH 4.5, 200 mM ammonium sulfate, and 11 to 14% (wt/vol) polyethylene glycol (PEG) 4000.…”