Crystal structures and small-angle x-ray scattering analysis of UDP-galactopyranose mutase from the pathogenic fungus Aspergillus fumigatus.

UDP-galactopyranose mutase (UGM) is a flavoenzyme that catalyzes the conversion of UDP-galactopyranose to UDP-galactofuranose, which is a central reaction in galactofuranose biosynthesis. Galactofuranose has never been found in humans but is an essential building block of the cell wall and extracellular matrix of many bacteria, fungi, and protozoa. The importance of UGM for the viability of many pathogens and its absence in humans make UGM a potential drug target. Here we report the first crystal structures and small-angle x-ray scattering data for UGM from the fungus Aspergillus fumigatus, the causative agent of aspergillosis. The structures reveal that Aspergillus UGM has several extra secondary and tertiary structural elements that are not found in bacterial UGMs yet are important for substrate recognition and oligomerization. Small-angle x-ray scattering data show that Aspergillus UGM forms a tetramer in solution, which is unprecedented for UGMs. The binding of UDP or the substrate induces profound conformational changes in the enzyme. Two loops on opposite sides of the active site move toward each other by over 10 Å to cover the substrate and create a closed active site. The degree of substrate-induced conformational change exceeds that of bacterial UGMs and is a direct consequence of the unique quaternary structure of Aspergillus UGM. Galactopyranose binds at the re face of the FAD isoalloxazine with the anomeric carbon atom poised for nucleophilic attack by the FAD N5 atom. The structural data provide new insight into substrate recognition and the catalytic mechanism and thus will aid inhibitor design.

Three crystal forms of the bifunctional enzyme proline utilization A (PutA) from Bradyrhizobium japonicum.

Proline utilization A proteins (PutAs) are large (1000-1300 residues) membrane-associated bifunctional flavoenzymes that catalyze the two-step oxidation of proline to glutamate by the sequential action of proline dehydrogenase and Delta(1)-pyrroline-5-carboxylate dehydrogenase domains. Here, the first successful crystallization efforts for a PutA protein are described. Three crystal forms of PutA from Bradyrhizobium japonicum are reported: apparent tetragonal, hexagonal and centered monoclinic. The apparent tetragonal and hexagonal crystals were grown in the presence of PEG 3350 and sodium formate near pH 7. The apparent tetragonal form diffracted to 2.7 A resolution and exhibited pseudo-merohedral twinning such that the true space group is P2(1)2(1)2(1) with four molecules in the asymmetric unit. The hexagonal form diffracted to 2.3 A resolution and belonged to space group P6(2)22 with one molecule in the asymmetric unit. Centered monoclinic crystals were grown in ammonium sulfate, diffracted to 2.3 A resolution and had two molecules in the asymmetric unit. Removing the histidine tag was important in order to obtain the C2 crystal form.

Crystallization of Phi29 spindle-shaped nano-bar anti-receptor with glycosidase domain.

Bacteriophage phi29 is a small, well-characterized dsDNA virus that infects Bacillus subtilis. The anti-receptor of phi29 consists of oligomers of the 854-residue protein gp12 and plays an essential role in infection initiation by binding to the receptor on the host cell surface. Oligomers of gp12 exhibit a narrow spindle-shaped configuration 15 nm in length as revealed by electron microscopy and thus are potentially useful nanoscale tools, building blocks, or motor arms. To understand the mechanism of viral infection initiation and to provide a basis for engineering recombinant gp12 for nanotechnology applications, we have initiated structural and bioinformatics studies of gp12. We report here the growth of crystals of gp12 that diffract to 3.0 A resolution. The space group is P3(1)21 or P3(2)21 with unit cell lengths of a = 84.4 A and c = 167.6 A. The asymmetric unit is predicted to contain one gp12 molecule and 32% solvent (VM = 1.8 A3/Da). Domain boundary analysis revealed that gp12 may harbor three domains besides a 24 residue auto-cleave region. The N-terminal half of gp12 contains a domain with about 400 residues that held 44% sequence identity to endopolygalacturonase, a fungal glycosyl hydrolase that catalyzes hydrolysis of the polygalacturonic acid alpha1-4 glycosidic linkage found in plant cell walls. Interestingly, the cell wall of Bacillus subtilis contains a polysaccharide component made from two sugar monomers, N-acetylmuramic acid and N-acetylglucosamine, which resemble alpha-galacturonic acid in that they possess a six-membered pyranose ring. Hence, polygalacturonic acid of plant cell walls and peptidoglycan of bacterial cell walls may offer a similar topography in relation to the polysaccharides. These results suggest a function for gp12 as a cell-wall degrading enzyme in addition to its role in recognition of the host receptor.