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Crystal structure of p-hydroxybenzoate hydroxylase complexed with its reaction product 3,4-dihydroxybenzoate

Crystals of the flavin-containing enzyme p-hydroxybenzoate hydroxylase (PHBHase) complexed with its reaction product were investigated in order to obtain insight into the catalytic cycle of this enzyme involving two substrates and two cofactors. PHBHase was crystallized initially with its substrate, p-hydroxybenzoate and the substrate was then converted into the product 3,4-dihydroxybenzoate by allowing the catalytic reaction to proceed in the crystals. In addition, crystals were soaked in mother liquor containing a high concentration of this product. Data up to 2.3 A (1 A = 0.1 nm) were collected by the oscillation method and the structure of the enzyme product complex was refined by alternate restrained least-squares procedures and model building by computer graphics techniques. A total of 273 solvent molecules could be located, four of them being presumably sulfate ions. The R-factor for 14,339 reflections between 6.0 A and 2.3 A is 19.3%. The 3-hydroxyl group of the product introduced by the enzyme is clearly visible in the electron density, showing unambiguously which carbon atom of the substrate is hydroxylated. A clear picture of the hydroxylation site is obtained. The plane of the product is rotated 21 degrees with respect to the plane of the substrate in the current model of enzyme-substrate complex. The 4-hydroxyl group of the product is hydrogen bonded to the hydroxyl group of Tyr201, its carboxyl group is interacting with the side-chains of Tyr222, Arg214 and Ser212, while the newly introduced 3-hydroxyl group makes a hydrogen bond with the backbone carbonyl oxygen of Pro293.

STRUCTURE OF PANULIRUS-INTERRUPTUS HEMOCYANIN AT 5 A RESOLUTION

The hemocyanin from the spiny lobster Panulirus interruptus, a hexamer with a molecular weight of approximately 540,000, was crystallized in space group P21 with two molecules in the unit cell and cell dimensions a = 119.8 A, b = 193.1 A, c = 122.2 A and β = 118.1 °. With screened precession photographs a three-dimensional set of reflections was collected up to 10 A resolution. Both the conventional and the fast rotation function programs were applied and gave results that were in excellent agreement with each other. The hemocyanin hexamer has 32 point group symmetry. Its 3-fold molecular axis runs approximately parallel to the crystallographic 2-fold screw axis. X-ray diffraction data to 5 A resolution were collected by the oscillation method. Rotation function studies with data between 7 and 5 A resolution confirmed the 10 A studies and, furthermore, showed that the rotation axes relating subunits within one hexameric molecule can be distinguished from the rotation axes relating subunits belonging to different hexamers in the unit cell. The local 3-fold axis in the hexamer makes an angle of about 6 ° with the crystallographic 2-fold screw axis. For a mercury and a platinum derivative three-dimensional data sets were collected to 5 A by the oscillation method. The difference Patterson of the platinum derivative could be solved. The eventual number of heavy-atom sites was 36 for the platinum derivative and 70 for the mercury derivative. From the well-occupied sites the point-group symmetry of the molecule could be established accurately. In addition, the centre of the hexamer could be located within 0.2 A. Protein phases were obtained from isomorphous as well as anomalous differences. A “best” electron density map calculated with these phases showed the shape of the hexameric molecule as well as the boundaries of the six subunits. Correlation coefficients between the densities of the subunits showed little variation, suggesting a random distribution of the different subunit types (Van Eerd & Folkerts, 1981) over the six positions in the hexamer. The subunits are positioned at the corner of an antiprism. When viewed along the 3-fold axis the hexamer is roughly hexagonal in shape, with a diameter of approximately 120 A. Viewed along one of the 2-fold axes the molecule is of rectangular shape with dimensions 95 A × 120 A. The subunit can be described as an ellipsoid of irregular shape with axes of 80 A, 55 A and 48 A. Each subunit makes extensive contacts with three other subunits in the hexamer and, possibly, a much weaker contact with a fourth subunit.

Preliminary crystallographic studies of triosephosphate isomerase from the blood parasite Trypanosoma brucei brucei.

Crystals of triosephosphate isomerase (EC 5.3.1.1) from Trypanosoma brucei brucei have been grown. These crystals diffract to at least 2 A, even after 60 hours of exposure to X-rays. The space group is P212121, with cell dimensions a = 112.4 A, b = 97.8 A, c = 48.0 A. There is one dimer per asymmetric unit.

Preliminary crystallographic studies of glycosomal glyceraldehyde phosphate dehydrogenase from Trypanosoma brucei brucei

Crystals of glyceraldehyde phosphate dehydrogenase from the glycosome of Trypanosoma brucei brucei have been grown, and a partial data set has been collected using synchrotron radiation. The crystals diffract initially to 2.3 A resolution. The space group is P2(1)2(1)2, with cell dimensions a = 135 A, b = 255 A, c = 115 A, so there are probably at least two tetramers in the asymmetric unit.