A combined solid-state NMR and synchrotron X-ray diffraction powder study on the structure of the antioxidant (+)-catechin 4.5-hydrate.

Analyses combining X-ray powder diffraction (XRD) and solid-state NMR (SSNMR) data can now provide crystal structures in challenging powders that are inaccessible by traditional methods. The flavonoid catechin is an ideal candidate for these methods, as it has eluded crystallographic characterization despite extensive study. Catechin was first described nearly two centuries ago, and its powders exhibit numerous levels of hydration. Here, synchrotron XRD data provide all heavy-atom positions in (+)-catechin 4.5-hydrate and establish the space group as C2. SSNMR data ((13)C tensor and (1)H/(13)C correlation) complete the conformation by providing catechin's five OH hydrogen orientations. Since 1903, this phase has been erroneously identified as a 4.0 hydrate, but XRD and density data establish that this discrepancy is due to the facile loss of the water molecule located at a Wyckoff special position in the unit cell. A final improvement to heavy-atom positions is provided by a geometry optimization of bond lengths and valence angles with XRD torsion angles held constant. The structural enhancement in this final structure is confirmed by the significantly improved fit of computed (13)C tensors to experimental data.

Application of molecular replacement to protein powder data from image plates.

Macromolecular structures can be solved via molecular replacement from powder diffraction data collected not only on multi-analyzer diffractometers but also on image plates. Diffraction peaks recorded on image plates are generally broader than those collected using an array of crystal analyzer detectors, but the image-plate data often allow the use of powder data to lower d-spacings. Owing to the high incidence of overlaps in powder patterns, which is especially evident for larger structures, a multi-pattern Pawley refinement is necessary in order to distinguish intensity peaks. This work utilized various salt concentrations to produce small lattice distortions, which resulted in shifts of Bragg peak positions, in a suite of five powder patterns. Using reflection structure factors obtained from this combined refinement, the structure of hen egg-white lysozyme was determined by molecular replacement using the 60% identical human lysozyme (PDB code 1lz1) as the search model. This work also expands upon previous work by presenting a full-scale multi-species analysis combined with an investigation of the sensitivity with regard to discrimination between incorrect fold types. To test the limits of this technique, extension to higher molecular-weight structures is ongoing.

Mechanism of Substrate and Inhibitor Binding of Rhodobacter capsulatus Xanthine Dehydrogenase.

Rhodobacter capsulatus xanthine dehydrogenase (XDH) is an (alphabeta)(2) heterotetrameric cytoplasmic enzyme that resembles eukaryotic xanthine oxidoreductases in respect to both amino acid sequence and structural fold. To obtain a detailed understanding of the mechanism of substrate and inhibitor binding at the active site, we solved crystal structures of R. capsulatus XDH in the presence of its substrates hypoxanthine, xanthine, and the inhibitor pterin-6-aldehyde using either the inactive desulfo form of the enzyme or an active site mutant (E(B)232Q) to prevent substrate turnover. The hypoxanthine- and xanthine-bound structures reveal the orientation of both substrates at the active site and show the importance of residue Glu(B)-232 for substrate positioning. The oxygen atom at the C-6 position of both substrates is oriented toward Arg(B)-310 in the active site. Thus the substrates bind in an orientation opposite to the one seen in the structure of the reduced enzyme with the inhibitor oxypurinol. The tightness of the substrates in the active site suggests that the intermediate products must exit the binding pocket to allow first the attack of the C-2, followed by oxidation of the C-8 atom to form the final product uric acid. Structural studies of pterin-6-aldehyde, a potent inhibitor of R. capsulatus XDH, contribute further to the understanding of the relative positioning of inhibitors and substrates in the binding pocket. Steady state kinetics reveal a competitive inhibition pattern with a K(i) of 103.57 +/- 18.96 nm for pterin-6-aldehyde.