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Dihydrodipicolinate synthase (DHDPS) is an oligomeric enzyme that catalyzes the first committed step of the lysine-biosynthesis pathway in plants and bacteria, which yields essential building blocks for cell-wall and protein synthesis. DHDPS is therefore of interest to drug-discovery research as well as to studies that probe the importance of quaternary structure to protein function, stability and dynamics. Accordingly, DHDPS from the psychrophilic (cold-dwelling) organism Shewanella benthica ( Sb-DHDPS) was cloned, expressed, purified and crystallized. The best crystals of Sb-DHDPS were grown in 200 m M ammonium sulfate, 100 m M bis-tris pH 5.0-6.0, 23-26%( w/ v) PEG 3350, 0.02%( w/ v) sodium azide and diffracted to beyond 2.5 Å resolution. Processing of diffraction data to 2.5 Å resolution resulted in a unit cell with space group P212121 and dimensions a = 73.1, b = 84.0, c = 143.7 Å. These studies of the first DHDPS enzyme to be characterized from a bacterial psychrophile will provide insight into the molecular evolution of enzyme structure and dynamics. [ABSTRACT FROM AUTHOR]

Dihydrodipicolinate synthase (DHDPS; EC 4.2.1.52) catalyzes the rate-limiting step in the ( S)-lysine biosynthesis pathway of bacteria and plants. Here, the cloning of the DHDPS gene from a clinical isolate of Streptococcus pneumoniae (OXC141 strain) and the strategy used to express, purify and crystallize the recombinant enzyme are described. Diffracting crystals were grown in high-molecular-weight PEG precipitants using the hanging-drop vapour-diffusion method. The best crystal, from which data were collected, diffracted to beyond 2.0 Å resolution. Initially, the crystals were thought to belong to space group P42212, with unit-cell parameters a = 105.5, b = 105.5, c = 62.4 Å. However, the R factors remained high following initial processing of the data. It was subsequently shown that the data set was twinned and it was thus reprocessed in space group P2, resulting in a significant reduction in the R factors. Determination of the structure will provide insight into the design of novel antimicrobial agents targeting this important enzyme from S. pneumoniae. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

In this paper, the crystallization and preliminary X-ray diffraction analysis to near-atomic resolution of DHDPS from Clostridium botulinum crystallized in the presence of its substrate pyruvate are presented. The enzyme crystallized in a number of forms using a variety of PEG precipitants, with the best crystal diffracting to 1.2 Å resolution and belonging to space group C2, in contrast to the unbound form, which had trigonal symmetry. The unit-cell parameters were a = 143.4, b = 54.8, c = 94.3 Å, β = 126.3°. The crystal volume per protein weight ( VM) was 2.3 Å3 Da−1 (based on the presence of two monomers in the asymmetric unit), with an estimated solvent content of 46%. The high-resolution structure of the pyruvate-bound form of C. botulinum DHDPS will provide insight into the function and stability of this essential bacterial enzyme. [ABSTRACT FROM AUTHOR]