E. Howard

Production of crystals of human aldose reductase with very high resolution diffraction

As the action of human aldose reductase (hAR) is thought to be linked to the pathogenesis of diabetic complications, much effort has been directed towards the analysis of the catalytic mechanism and the development of specific inhibitors. Here, the crystallization of recombinant hAR with its cofactor NADP+ at 277 K in the presence of the precipitating agent PEG 6000 is reported. The crystals diffract to high resolution (1.1 A) and belong to the P21 space group with unit-cell parameters a = 49.97, b = 67.14, c = 48. 02 A, beta = 92.2 degrees with one molecule per asymmetric unit. Seleno-substituted hAR crystals were also produced and diffract to 1. 7 A on a conventional X-ray source.

The structure of human aldose reductase bound to the inhibitor IDD384.

The crystallographic structure of the complex between human aldose reductase (AR2) and one of its inhibitors, IDD384, has been solved at 1.7 A resolution from crystals obtained at pH 5.0. This structure shows that the binding of the inhibitors hydrophilic head to the catalytic residues Tyr48 and His110 differs from that found previously with porcine AR2. The difference is attributed to a change in the protonation state of the inhibitor (pK(a) = 4.52) when soaked with crystals of human (at pH 5.0) or pig lens AR2 (at pH 6.2). This work demonstrates how strongly the detailed binding of the inhibitors polar head depends on its protonation state.

Discovery of (R)-2-Amino-6-borono-2-(2-(piperidin-1-yl)ethyl)hexanoic Acid and Congeners As Highly Potent Inhibitors of Human Arginases I and II for Treatment of Myocardial Reperfusion Injury.

Recent efforts to identify treatments for myocardial ischemia reperfusion injury have resulted in the discovery of a novel series of highly potent α,α-disubstituted amino acid-based arginase inhibitors. The lead candidate, (R)-2-amino-6-borono-2-(2-(piperidin-1-yl)ethyl)hexanoic acid, compound 9, inhibits human arginases I and II with IC50s of 223 and 509 nM, respectively, and is active in a recombinant cellular assay overexpressing human arginase I (CHO cells). It is 28% orally bioavailable and significantly reduces the infarct size in a rat model of myocardial ischemia/reperfusion injury. Herein, we report the design, synthesis, and structure-activity relationships (SAR) for this novel series of inhibitors along with pharmacokinetic and in vivo efficacy data for compound 9 and X-ray crystallography data for selected lead compounds cocrystallized with arginases I and II.

High-resolution neutron and X-ray diffraction room-temperature studies of an H-FABP-oleic acid complex: study of the internal water cluster and ligand binding by a transferred multipolar electron-density distribution

Neutron and high-resolution X-ray crystallography were used to determine fully the structure of the internal water cluster in H-FABP. Analysis of the orientation and electrostatic properties of the water molecules showed significant alignment of the permanent dipoles of the water molecules with the protein electrostatic field.

New insights into the enzymatic mechanism of human chitotriosidase (CHIT1) catalytic domain by atomic resolution X-ray diffraction and hybrid QM/MM.

Chitotriosidase (CHIT1) is a human chitinase belonging to the highly conserved glycosyl hydrolase family 18 (GH18). GH18 enzymes hydrolyze chitin, an N-acetylglucosamine polymer synthesized by lower organisms for structural purposes. Recently, CHIT1 has attracted attention owing to its upregulation in immune-system disorders and as a marker of Gaucher disease. The 39u2005kDa catalytic domain shows a conserved cluster of three acidic residues, Glu140, Asp138 and Asp136, involved in the hydrolysis reaction. Under an excess concentration of substrate, CHIT1 and other homologues perform an additional activity, transglycosylation. To understand the catalytic mechanism of GH18 chitinases and the dual enzymatic activity, the structure and mechanism of CHIT1 were analyzed in detail. The resolution of the crystals of the catalytic domain was improved from 1.65u2005Å (PDB entry 1waw) to 0.95-1.10u2005Å for the apo and pseudo-apo forms and the complex with chitobiose, allowing the determination of the protonation states within the active site. This information was extended by hybrid quantum mechanics/molecular mechanics (QM/MM) calculations. The results suggest a new mechanism involving changes in the conformation and protonation state of the catalytic triad, as well as a new role for Tyr27, providing new insights into the hydrolysis and transglycosylation activities.

Joint neutron and X-ray diffraction studies at 293 K of antifreeze protein

Human thioredoxin1 (hTRX1) is a small 12-kD oxidoreductase enzyme consisting of 105 amino acids and containing a dithiol/ disulfide active site with multiple cellular functions. This enzyme has activity as a cellular reductase by a dithiol-disulfide exchange reaction using two cysteine residues (Cys32 and Cys35) in the conserved active site sequence. Apart from the two cysteines, there are three additional conserved cysteines, Cys62, Cys69, and Cys73 in the mammalian TRX, which have not been known to their biological functions. Although it has been identified that the Cys73 residue is involved in dimerization of hTRX via an intermolecular disulfide bond formation between Cys73 of each monomer in the oxidized state, biological function of the Cys62 and Cys69 residues in the nonactive remain to be fully elucidated. In the previous paper, researchers proposed that the formation of a disulfide bond between Cys62 and Cys69 could give a way to transiently inhibit hTRX activity for redox signaling or oxidative stress. Furthermore, they proposed a model structure of the non-active site disulfide in the hTRX. Here, we present the high-resolution crystal structure of fully oxidized hTRX1, which shows an intramolecular disulfide bond between Cys62 and Cys69. The disulfide bond formation disengages a helix proximal to the active site and results in a conformational change of the hTRX enzyme, providing a structural basis for understanding the regulation mechanism of redox signaling or oxidative stress.

Inhibitor binding to aldose reductase studied at subatomic resolution

Resolution Alberto Podjarny, Andre Mitschler, Isabelle Hazemann, Tania Petrova, Federico Ruiz, Eduardo Howard, Connie Darmanin, Roland Chung, Thomas R. Schneider, Ruslan Sanishvili, Clemens Schulze-Briesse, Takashi Tomizaki, Michael Van Zandt, Mitsuru Oka, Andrzej Joachimiak, Ossama El-Kabbani, IGBMC, CNRS, Illkirch, France. Department of Medicinal Chemistry, Monash U., Australia. FIRC Institute of Molecular Oncology, Milan, Italy. SBC, APS, Argonne, Illinois, USA. SLS, PSI, Villigen, Switzerland. IDD, Branford, CT, USA. Sanwa Kagaku Kenkyusyo Ltd., Japan. E-mail: podjarny@igbmc.u-strasbg.fr