h-AR (Enzyme Commission 1.1.1.21) is a NADPH-dependent enzyme that reduces a wide range of substrates, such as aldehydes, aldoses, and corticosteroids. As it reduces D-glucose into D-sorbitol, it is believed to cause severe degenerative complications of diabetes (1). The basic catalytic reaction involves a hydride transfer from C4 of the nicotinamide ring of NADPH and a proton donation from the enzyme (2) - however, the identity of the residue donating the proton, and by what mechanism this occurs has proved controversial. Using a combination of single-crystal X-ray data [0.66 Å, 100K (3, 4); 0.80 Å, 15K; 1.75 Å, 293K (4)], neutron Laue data [2.2 Å, 293K (4, 5)], and quantum mechanical modeling for either hydrogenated or perdeuterated ternary h-AR/NADP+/IDD594 complexes, it was possible to unveil the internal organization and mobility of the hydrogen bond network that defines the properties of the catalytic engine (4). The results suggest this enzyme overcomes the difficulty of simultaneously satisfying the requirements of being an effective catalyst and a promiscuous one by using a distal proton donor (Asp-43–Lys-77 pair) acting on a flexible final proton carrier (Tyr-48), capable of accommodating different substrates. The direct observation of the properties of the proton network, extending from Asp-43 to IDD594, were only possible by combining the information from X-ray and neutron diffraction data and thus emphasizes the importance of these complementary techniques in structural biology.

References

(*Those in green are of particular interest)

1. Yabe-Nishimura, C. (1998) Pharmacological Review 50, 21-33.

2. Wermuth, B. (1985) Enzymology of Carbonyl Metabolism 2: Aldehyde Dehydrogenase, Aldo-Keto Reductase, and Alcohol Dehydrogenase (Alan R. Liss Inc., New York, New York).

3. Howard, E. I., Sanishvili, R., Cachau, R. E., Mitschler, A., Chevrier, B., Barth, P., Lamour, V., Van Zandt, M., Sibley, E., Bon, C., Moras, D., Schneider, T. R., Joachimiak, A. & Podjarny, A. D. (2004) Proteins: Struct Funct Genet. 55, 792-804.

4*. Blakeley, M. P., Ruiz, F., Cachau, R., Hazemann, I., Meilleur, F., Mitschler, A., Ginell, S., Afonine, P., Ventura, O. N., Cousido-Siah, A., Haertlein, M., Joachimiak, A., Myles, D. A. A. & Podjarny, A. D. (2008) Proceedings Natiaonal Academy of Sciences, USA. 105(6), 1844-1848.

5*. Hazemann, I., Dauvergne, M.T., Blakeley, M. P., Meilleur, F., Haertlein, M. Van Dorsselaer, A., Mitschler, A., Myles, D. A. A. & Podjarny, A. D. (2005) Acta Cryst. D61, 1413-1417.

Concanavalin A (Con A) is a saccharide-binding protein belonging to the legume lectin family. The monomer of Con A is dominated by an extensive β-sheet arrangement and contains two metal-binding sites (a transition metal-binding site and a Ca²⁺ binding site), both of which must be occupied for the protein to bind saccharide (1). The Mn²⁺ site has a slightly distorted octahedral geometry in which it is coordinated to Asp-10Oδ1, Asp-19Oδ1, Glu-8Oε1, the imidazole Nε of His-24, and two water molecules. The Ca²⁺ site has a pseudo octahedral geometry in which it is coordinated to Asp-10 Oδ1 and Oδ2, Asp-19 Oδ2, Tyr-12 O, Asn-14 Oδ1, and two water molecules. Previously on LADI-I, the neutron structure of the D₂O soaked, saccharide-free form of Con A was determined to 2.4 Å at room temperature (2). The 293K study showed the nature of the metal ligand regions of Con A, specifically revealing details of the orientation of the four bound waters’ D atoms, which were, as expected, pointing away from the Mn²⁺ and Ca²⁺ ions. The study also allowed the positions and orientations of the bound water D atoms at the saccharide-binding site to be modeled, although the clarity of the nuclear density at the individual waters was poor, i.e., not revealing them singly but rather as a cluster. By collecting neutron data at cryo temperatures, the dynamic disorder within a protein crystal is reduced, which may lead to improved definition of the nuclear density. Information on the orientation of such waters to the approach of the ligand is important for more complete thermodynamics and modeling studies (3). It has proved possible to cryo-cool very large Con A protein crystals (>1.5mm³) suitable for high-resolution neutron and X-ray structure analysis. The 15K neutron crystal structure of the saccharide-free form of Con A and its bound water, including 167 intact D₂O molecules was determined to 2.5 Å resolution, along with the 1.65 Å X-ray structure of an identical crystal at 100 K (4). Comparison with the 293 K neutron structure shows that the bound water molecules are better ordered and have lower average B factors than those at room temperature. Overall, twice as many bound waters (as D₂O) are identified at 15 K than at 293 K. We note that alteration of bound water orientations occurs between 293 and 15 K; such changes could be important more generally in protein crystal structure analysis and ligand design. Methodologically, this successful neutron cryo protein structure refinement opens up categories of neutron protein crystallography, including freeze-trapped structures and cryo to room temperature comparisons.

Urate oxidase (Uox) is an enzyme which catalyses the oxidation of uric acid to allantoin, an inactive and soluble metabolite. It is inactivated in humans and higher primates. Urate oxidase is used as a protein drug to reduce toxic uric acid accumulation and to resolve the hyperuricaemic disorders occurring during chemotherapy. The mechanism, which converts uric acid into allantoin, is composed of many steps (1, 2). The first step is catalyzed by urate oxidase, which oxidizes uric acid to an intermediate compound. The intermediate compound is then degraded to allantoin through a number of chemical steps. The recent X-ray structures of urate oxidase-inhibitor complexes (3 - 5) have provided several new details towards understanding the mechanism. However, details of the positions of the H atoms within the active site, critical for a complete understanding of the mechanism are still unknown. Thus, in order to locate the H-atoms within the active site and provide key insights into the proton shuttle involved in the urate oxidase-catalyzed oxidation of uric acid, neutron diffraction data has been collected on the LADI-I instrument (6). Neutron Laue diffraction data were collected to 2.1 Å from a crystal (volume 1.8 mm³, grown in D₂O) of a urate oxidase/8-azaxanthin complex. The neutron diffraction data collected are of high quality and structural refinement is currently underway. In comparison with other neutron protein crystallography studies to date, urate oxidase has one of the largest primitive unit-cell volumes (space group I222, unit-cell parameters a = 80, b = 96, c = 106 Å) and molecular weights (135 kDa for the homotetramer) so far successfully studied with neutrons.