Cloning, expression, crystallization and preliminary X-ray crystallographic analysis of beta-ketoacyl-ACP synthase III (FabH) from Xanthomonas oryzae pv. oryzae.

The bacterial beta-ketoacyl-ACP synthase III (KASIII) encoded by the gene fabH (Xoo4209) from Xanthomonas oryzae pv. oryzae, a plant pathogen, is an important enzyme in the elongation steps of fatty-acid biosynthesis. It is expected to be one of the enzymes responsible for bacterial blight (BB), a serious disease that results in huge production losses of rice. As it represents an important target for the development of new antibacterial drugs against BB, determination of the crystal structure of the KAS III enzyme is essential in order to understand its reaction mechanism. In order to analyze the structure and function of KAS III, the fabH (Xoo4209) gene was cloned and the enzyme was expressed and purified. The KASIII crystal diffracted to 2.05 A resolution and belonged to the orthorhombic space group P2(1)2(1)2, with unit-cell parameters a = 69.8, b = 79.5, c = 62.3 A. The unit-cell volume of the crystal is compatible with the presence of a single monomer in the asymmetric unit, with a corresponding Matthews coefficient V(M) of 2.27 A(3) Da(-1) and a solvent content of 45.8%.

Cloning, expression, crystallization and preliminary X-ray crystallographic analysis of 3-dehydroquinate synthase, Xoo1243, from Xanthomonas oryzae pv. oryzae.

The disease bacterial blight results in serious production losses of rice in Asian countries. The aroB gene encoding dehydroquinate synthase (DHQS), which is a potential antibiotic target, was identified from the plant-pathogenic bacterium Xanthomonas oryzae pv. oryzae (Xoo). DHQS plays an essential role in the synthesis of aromatic compounds in the shikimate pathway. The aroB gene (Xoo1243) was cloned from Xoo and the corresponding DHQS protein was subsequently overexpressed in Escherichia coli. The purified protein was crystallized using the hanging-drop vapour-diffusion method and yielded crystals that diffracted to 2.5 A resolution. The crystals belonged to the tetragonal space group P4(3)2(1)2, with unit-cell parameters a = b = 118.2, c = 98.2 A. According to a Matthews coefficient calculation, the crystal contained two molecules in the asymmetric unit, with a corresponding V(M) of 2.06 A(3) Da(-1) and a solvent content of 40.4%.

Expression, crystallization and preliminary X-ray crystallographic analysis of peptide deformylase from Xanthomonas oryzae pv. oryzae.

Peptide deformylase (PDF) catalyzes the removal of the N-formyl group from the N-terminus of newly synthesized polypeptides; this process is crucial for cell survival. As it is an antibacterial drug target against Xanthomonas oryzae pv. oryzae (Xoo), PDF from Xoo was cloned, expressed, purified and crystallized. Native PDF crystals diffracted to 2.7 A resolution and belonged to the hexagonal space group P6(1)22, with unit-cell parameters a = b = 59.0, c = 266.3 A. One monomer is present in the asymmetric unit, with a corresponding crystal volume per protein weight of 3.50 A(3) Da(-1) and a solvent content of 64.9%.

Expression, crystallization and preliminary X-ray crystallographic analysis of XometC, a cystathionine gamma-lyase-like protein from Xanthomonas oryzae pv. oryzae.

Xanthomonas oryzae pv. oryzae (Xoo) causes bacterial blight of rice (Oryza sativa L.), one of the most devastating diseases of rice in most rice-growing countries. XometC, a cystathionine gamma-lyase (CGL) like protein that is an antibacterial drug-target protein against Xoo, was cloned, expressed, purified and crystallized. CGL catalyzes the second step in the reverse-transsulfuration pathway, which is essential for the metabolic interconversion of the sulfur-containing amino acids cysteine and methionine. Crystals of two different shapes, plate-shaped and pyramid-shaped, diffracted to 2.9 and 3.2 A resolution and belonged to the primitive orthogonal space group P2(1)2(1)2(1) and the tetragonal space group P4(1) (or P4(3)), with unit-cell parameters a = 73.0, b = 144.9, c = 152.3 A and a = b = 78.2, c = 300.7 A, respectively. For the P2(1)2(1)2(1) crystals, three or four monomers exist in the asymmetric unit with a corresponding V(M) of 3.02 or 2.26 A(3) Da(-1) and a solvent content of 59.3 or 45.7%. For the P4(1) (or P4(3)) crystals, four or five monomers exist in the asymmetric unit with a corresponding V(M) of 2.59 or 2.09 A(3) Da(-1) and a solvent content of 52.5 or 40.6%.