UDP-galactopyranose mutase has a novel structure and mechanism.

Uridine diphosphogalactofuranose (UDP-Galf ) is the precursor of the d-galactofuranose (Galf ) residues found in bacterial and parasitic cell walls, including those of many pathogens, such as Mycobacterium tuberculosis and Trypanosoma cruzi. UDP-Galf is made from UDP-galactopyranose (UDP-Galp) by the enzyme UDP-galactopyranose mutase (mutase). The mutase enzyme is essential for the viability of mycobacteria and is not found in humans, making it a viable therapeutic target. The mechanism by which mutase achieves the unprecedented ring contraction of a nonreducing sugar is unclear. We have solved the crystal structure of Escherichia coli mutase to 2.4 A resolution. The novel structure shows that the flavin nucleotide is located in a cleft lined with conserved residues. Site-directed mutagenesis studies indicate that this cleft contains the active site, with the sugar ring of the substrate UDP-galactose adjacent to the exposed isoalloxazine ring of FAD. Assay results establish that the enzyme is active only when flavin is reduced. We conclude that mutase most likely functions by transient reduction of substrate.

Molecular placement of experimental electron density: a case study on UDP-galactopyranose mutase.

The structure of UDP-galactopyranose mutase, the enzyme responsible for the conversion of UDP-galactopyranose to UDP-galactofuranose, has been solved. The structure solution required the use of two crystal forms and a selenomethionine variant. Crystal form P2(1) was used to collect a complete MAD data set, a native data set and a single-wavelength non-isomorphous selenomethionine data set. A starting set of MAD phases was then improved by non-crystallographic averaging and cross-crystal averaging of all P2(1) data. The initial maps were of such low quality that transformation matrices between cells could not be determined. It was therefore assumed that although there were large changes in unit-cell parameters, the molecule occupied the same position in each cell. This starting assumption was allowed to refine during the averaging procedure and did so satisfactorily. Despite a visible increase in the quality of the map allowing some secondary-structural elements to be located, the overall structure could not be traced and refined. The rediscovery of the second crystal form, P2(1)2(1)2(1), allowed the collection of a native data set to 2.4 A. Molecular placement of electron density was used to determine the relationship between the two unit cells. In this study, only the already averaged P2(1) experimental density could be placed in the P2(1)2(1)2(1) map. Less extensively density-modified maps did not give a clear solution. The study suggests even poor non-isomorphous data can be used to significantly improve map quality. The relationship between P2(1) and P2(1)2(1)2(1) could then be used in a final round of cross-crystal averaging to generate phases. The resulting map was easily traced and the structure has been refined. The structure sheds important light on a novel mechanism and is also a therapeutic target in the treatment of tuberculosis.

Overexpression, purification, crystallization and data collection of 3-methylaspartase from Clostridium tetanomorphum.

3-Methylaspartase (E.C. 4.3.1.2) catalyses the reversible anti elimination of ammonia from L-threo-(2S,3S)-3-methylaspartic acid to give mesaconic acid as well as a slower syn elimination from the (2S,3R)-epimer, L-erythro-3-methylaspartic acid. The anti-elimination reaction occurs in the second step of the catabolic pathway for glutamic acid in Clostridium tetanomorphum. The reverse reaction is of particular interest because the addition of ammonia to substituted fumaric acids is highly stereoselective and gives highly functionalized amino acids. The mechanism of the transformation is unusual and of considerable interest. 3-Methylaspartase from C. tetanomorphum has been overexpressed and purified from Escherichia coli. Crystals of the enzyme have been obtained by sitting-drop vapour diffusion. Two native data sets have been collected, one in-house on a rotating-anode generator to 3.2 A and one at the European Synchrotron Radiation Facility to 2.0 A. A 2.1 A data set has been collected on a crystal of selenomethionine protein. Combining the data sets identify the space group as P2(1)2(1)2, with unit-cell parameters a = 110.3, b = 109.9, c = 67.2 A, alpha = beta = gamma = 90 degrees. The asymmetric unit contains two monomers with 42% solvent. A self-rotation function indicates the presence of a twofold axis, consistent with a biological dimer.

Initiating a crystallographic study of UDP-galactopyranose mutase from Escherichia coli.

UDP-galactopyranose mutase, the enzyme responsible for the conversion of UDP-galactopyranose to UDP-galactofuranose, has been crystallized in a form suitable for X-ray diffraction studies. UDP-galactofuranose is a key component of mycobacterial cell walls. Crystals of both the native protein and a selenomethionine variant have been grown by the vapour-diffusion method in hanging drops, and diffract to beyond 3.0 A using synchrotron radiation. Equilibration was against a solution of 20%(w/v) polyethylene glycol (4K), 12%(v/v) 2--propanol, 0.1 M HEPES pH 7.6 at 293.5 K. Crystals grow as thin plates of dimensions 0.4 x 0.2 x approximately 0.02 mm. They are monoclinic [corrected], space group P21, with unit-cell dimensions a = 71. 12, b = 58.42, c = 96.38 A, beta = 96.38 degrees. 92% (native) and 94% (selenomethionine) complete data sets have been recorded to 2.9 A (Rmerge = 5.0%) and 3.0 A (Rmerge = 6.9%), respectively. The Matthews coefficient is 2.35 A3 Da-1 for a dimer in the asymmetric unit, the solvent content being 47%. Diffraction data have also been recorded on a putative platinum derivative to 3.5 A.

Purification, crystallization and preliminary structural studies of dTDP-6-deoxy-D-xylo-4-hexulose 3,5-epimerase (RmlC), the third enzyme of the dTDP-L-rhamnose synthesis pathway, from Salmonella enterica serovar typhimurium.

L-Rhamnose is an essential component of the cell wall of many pathogenic bacteria. Its precusor, dTDP-L-rhamnose, is synthesized from alpha-D-glucose-1-phosphate and dTTP via a pathway requiring four distinct enzymes: RmlA, RmlB, RmlC and RmlD. RmlC was overexpressed in Escherichia coli. The recombinant protein was purified by a two-step protocol involving anion-exchange and hydrophobic chromatography. Dynamic light-scattering experiments indicated that the recombinant protein is monodisperse. Crystals were obtained using the sitting-drop vapour-diffusion method with ammonium sulfate as precipitant. Diffraction data were collected on a frozen crystal to a resolution of 2.17 A. The crystal belongs to either space group P3121 or P3221, with unit-cell parameters a = b = 71.56, c = 183.53 A and alpha = beta = 90, gamma = 120 degrees.

Crystallization of succinylated concanavalin A bound to a synthetic bivalent ligand and preliminary structural analysis.

Crystals have been obtained of succinylated concanavalin A complexed to a novel bidentate synthetic ligand. The crystals are the first example of a lectin with a synthetic multivalent ligand and the first report of crystallization of succinylated concanavalin A. The crystals were obtained by sitting-drop vapour diffusion equilibrating with a solution of 20% polyethylene glycol, pH 5, 293. 5 K. Crystals are orthorhombic, belonging to space group C2221 with unit-cell dimensions of a = 99.1, b = 127.4, c = 118.9 A. The asymmetric unit contains a dimer, with over 65% of the volume occupied by water. The ligand cross links concanavalin A monomers. Succinylated concanavalin A is known to be a dimer in solution, yet it is found as the typical concanavalin A tetramer in the crystal. The contacts holding together the tetramer appear extensive and suggest that a fine balance between dimer and tetramers exists. Data to 2.65 A have been collected and the structure determined by the molecular replacement method.

Serial neurologic examination of hyperactive children.

Forty-four of 102 children undergoing pharmacologic treatment of hyperactivity received serial neurologic examinations on five separate occasions. The responses to three of the "soft" neurologic signs administered were analyzed: heel gait, toe gait, and diadochokinesis. A high degree of variability of response within individuals was documented. There was no evidence of interaction between treatment and the subjects' responses. The degree of variability was such that to have used these data as measures of "improvement" (i.e., drug effect) would have been misleading. The value of these signs for purposes of diagnosis or assessment of therapy is doubtful.

Side effects of dextroamphetamine therapy of hyperactive children.

Dextroamphetamine, prescribed in the treatment of hyperactive children, was associated with significant personality deterioration in five of 26 treated cases. Discontinuance of the drug and, in some cases, substitution of others was followed by lessened symptoms of disorganization or of toxicity. In general, children being treated with psychostimulants should be kept under careful observation for untoward reactions.