Bryan T. Eger

An Extremely Potent Inhibitor of Xanthine Oxidoreductase CRYSTAL STRUCTURE OF THE ENZYME-INHIBITOR COMPLEX AND MECHANISM OF INHIBITION

TEI-6720 (2-(3-cyano-4-isobutoxyphenyl)-4-methyl-5-thiazolecarboxylic acid) is an extremely potent inhibitor of xanthine oxidoreductase. Steady state kinetics measurements exhibit mixed type inhibition withK i and K i ′ values of 1.2 ± 0.05 × 10−10 m and 9 ± 0.05 × 10−10 m, respectively. Fluorescence-monitored titration experiments showed that TEI-6720 bound very tightly to both the active and the inactive desulfo-form of the enzyme. The dissociation constant determined for the desulfo-form was 2 ± 0.03 × 10−9 m; for the active form, the corresponding number was too low to allow accurate measurements. The crystal structure of the active sulfo-form of milk xanthine dehydrogenase complexed with TEI-6720 and determined at 2.8-Å resolution revealed the inhibitor molecule bound in a long, narrow channel leading to the molybdenum-pterin active site of the enzyme. It filled up most of the channel and the immediate environment of the cofactor, very effectively inhibiting the activity of the enzyme through the prevention of substrate binding. Although the inhibitor did not directly coordinate to the molybdenum ion, numerous hydrogen bonds as well as hydrophobic interactions with the protein matrix were observed, most of which are also used in substrate recognition.

Mammalian xanthine oxidoreductase – mechanism of transition from xanthine dehydrogenase to xanthine oxidase

Reactive oxygen species are generated by various biological systems, including NADPH oxidases, xanthine oxidoreductase, and mitochondrial respiratory enzymes, and contribute to many physiological and pathological phenomena. Mammalian xanthine dehydrogenase (XDH) can be converted to xanthine oxidase (XO), which produces both superoxide anion and hydrogen peroxide. Recent X‐ray crystallographic and site‐directed mutagenesis studies have revealed a highly sophisticated mechanism of conversion from XDH to XO, suggesting that the conversion is not a simple artefact, but rather has a function in mammalian organisms. Furthermore, this transition seems to involve a thermodynamic equilibrium between XDH and XO; disulfide bond formation or proteolysis can then lock the enzyme in the XO form. In this review, we focus on recent advances in our understanding of the mechanism of conversion from XDH to XO.

Mechanism of inhibition of xanthine oxidoreductase by allopurinol: crystal structure of reduced bovine milk xanthine oxidoreductase bound with oxipurinol.

Inhibitors of xanthine oxidoreductase block conversion of xanthine to uric acid and are therefore potentially useful for treatment of hyperuricemia or gout. We determined the crystal structure of reduced bovine milk xanthine oxidoreductase complexed with oxipurinol at 2.0 Å resolution. Clear electron density was observed between the N2 nitrogen of oxipurinol and the molybdenum atom of the molybdopterin cofactor, indicating that oxipurinol coordinated directly to molybdenum. Oxipurinol forms hydrogen bonds with glutamate802, arginine880, and glutamate1261, which have previously been shown to be essential for the enzyme reaction. We discuss possible differences in the hypouricemic effect of inhibitors, including allopurinol and newly developed inhibitors, based on their mode of binding in the crystal structures.

Unique amino acids cluster for switching from the dehydrogenase to oxidase form of xanthine oxidoreductase

In mammals, xanthine oxidoreductase is synthesized as a dehydrogenase (XDH) but can be readily converted to its oxidase form (XO) either by proteolysis or modification of cysteine residues. The crystal structures of bovine milk XDH and XO demonstrated that atoms in the highly charged active-site loop (Gln-423–Lys-433) around the FAD cofactor underwent large dislocations during the conversion, blocking the approach of the NAD+ substrate to FAD in the XO form as well as changing the electrostatic environment around FAD. Here we identify a unique cluster of amino acids that plays a dual role by forming the core of a relay system for the XDH/XO transition and by gating a solvent channel leading toward the FAD ring. A more detailed structural comparison and site-directed mutagenesis analysis experiments showed that Phe-549, Arg-335, Trp-336, and Arg-427 sit at the center of a relay system that transmits modifications of the linker peptide by cysteine oxidation or proteolytic cleavage to the active-site loop (Gln-423–Lys-433). The tight interactions of these residues are crucial in the stabilization of the XDH conformation and for keeping the solvent channel closed. Both oxidative and proteolytic generation of XO effectively leads to the removal of Phe-549 from the cluster causing a reorientation of the bulky side chain of Trp-336, which then in turn forces a dislocation of Arg-427, an amino acid located in the active-site loop. The conformational change also opens the gate for the solvent channel, making it easier for oxygen to reach the reduced FAD in XO.

Activators of Cylindrical Proteases as Antimicrobials: Identification and Development of Small Molecule Activators of ClpP Protease

ClpP is a cylindrical serine protease whose ability to degrade proteins is regulated by the unfoldase ATP-dependent chaperones. ClpP on its own can only degrade small peptides. Here, we used ClpP as a target in a high-throughput screen for compounds, which activate the protease and allow it to degrade larger proteins, hence, abolishing the specificity arising from the ATP-dependent chaperones. Our screen resulted in five distinct compounds, which we designate as Activators of Self-Compartmentalizing Proteases 1 to 5 (ACP1 to 5). The compounds are found to stabilize the ClpP double-ring structure. The ACP1 chemical structure was considered to have drug-like characteristics and was further optimized to give analogs with bactericidal activity. Hence, the ACPs represent classes of compounds that can activate ClpP and that can be developed as potential novel antibiotics.

Protein conformational gating of enzymatic activity in xanthine oxidoreductase

In mammals, xanthine oxidoreductase can exist as xanthine dehydrogenase (XDH) and xanthine oxidase (XO). The two enzymes possess common redox active cofactors, which form an electron transfer (ET) pathway terminated by a flavin cofactor. In spite of identical protein primary structures, the redox potential difference between XDH and XO for the flavin semiquinone/hydroquinone pair (E(sq/hq)) is ~170 mV, a striking difference. The former greatly prefers NAD(+) as ultimate substrate for ET from the iron-sulfur cluster FeS-II via flavin while the latter only accepts dioxygen. In XDH (without NAD(+)), however, the redox potential of the electron donor FeS-II is 180 mV higher than that for the acceptor flavin, yielding an energetically uphill ET. On the basis of new 1.65, 2.3, 1.9, and 2.2 Å resolution crystal structures for XDH, XO, the NAD(+)- and NADH-complexed XDH, E(sq/hq) were calculated to better understand how the enzyme activates an ET from FeS-II to flavin. The majority of the E(sq/hq) difference between XDH and XO originates from a conformational change in the loop at positions 423-433 near the flavin binding site, causing the differences in stability of the semiquinone state. There was no large conformational change observed in response to NAD(+) binding at XDH. Instead, the positive charge of the NAD(+) ring, deprotonation of Asp429, and capping of the bulk surface of the flavin by the NAD(+) molecule all contribute to altering E(sq/hq) upon NAD(+) binding to XDH.

Crystallogenesis of Membrane Proteins Mediated by Polymer-Bounded Lipid Nanodiscs

For some membrane proteins, detergent-mediated solubilization compromises protein stability and functionality, often impairing biophysical and structural analyses. Hence, membrane-protein structure determination is a continuing bottleneck in the field of protein crystallography. Here, as an alternative to approaches mediated by conventional detergents, we report the crystallogenesis of a recombinantly produced membrane protein that never left a lipid bilayer environment. We used styrene-maleic acid (SMA) copolymers to solubilize lipid-embedded proteins into SMA nanodiscs, purified these discs by affinity and size-exclusion chromatography, and transferred proteins into the lipidic cubic phase (LCP) for in meso crystallization. The 2.0-Å structure of an α-helical seven-transmembrane microbial rhodopsin thus obtained is of high quality and virtually identical to the 2.2-Å structure obtained from traditional detergent-based purification and subsequent LCP crystallization.

Crystal Structures of Urate Bound Form of Xanthine Oxidoreductase: Substrate Orientation and Structure of the Key Reaction Intermediate

Two contradictory models have been proposed for the binding mode of the substrate xanthine to and its activation mechanism by xanthine oxidoreductase. In an effort to distinguish between the two models, we determined the crystal structures of the urate complexes of the demolybdo-form of the D428A mutant of rat xanthine oxidoreductase at 1.7 Å and of the reduced bovine milk enzyme at 2.1 Å, the latter representing a reaction intermediate. The results clearly indicate the catalytically relevant binding mode of the substrate xanthine.

Purification, crystallization and preliminary X-ray diffraction studies of xanthine dehydrogenase and xanthine oxidase isolated from bovine milk.

Xanthine dehydrogenase catalyzes the oxidation of hypoxanthine to xanthine and the further oxidation of xanthine to uric acid. The enzyme is the target of the anti-gout drug allopurinol and its involvement in postischemic reperfusion injury is presently being defined. Each subunit of the homodimeric 290 kDa enzyme contains four cofactors: one Mo-pterin, two [2Fe-2S] clusters and one FAD. Both the dehydrogenase (XDH) and the proteolytically modified oxidase form (XO) of the enzyme from bovine milk have been crystallized. XO crystals belong to space group C222(1), with unit-cell parameters a = 116.3, b = 164.4, c = 153.2 A at room temperature and a = 117.8, b = 165.4, c = 154.5 A when flash-frozen. They allow data collection to 3.3 and 2.5 A, respectively. In addition, a data set was collected from frozen XDH crystals and processed to 2.1 A. These crystals belong to space group C2, with unit-cell parameters a = 169.9, b = 124.8, c = 148.6 A, beta = 90.9 degrees. The unit-cell volumes and Matthews parameters are similar for the two crystal forms. There is one monomer per asymmetric unit in the XO crystals and a complete native dimer per asymmetric unit in the XDH crystals.

Fixed target combined with spectral mapping: approaching 100% hit rates for serial crystallography.

The advent of ultrafast highly brilliant coherent X-ray free-electron laser sources has driven the development of novel structure-determination approaches for proteins, and promises visualization of protein dynamics on sub-picosecond timescales with full atomic resolution. Significant efforts are being applied to the development of sample-delivery systems that allow these unique sources to be most efficiently exploited for high-throughput serial femtosecond crystallography. Here, the next iteration of a fixed-target crystallography chip designed for rapid and reliable delivery of up to 11 259 protein crystals with high spatial precision is presented. An experimental scheme for predetermining the positions of crystals in the chip by means of in situ spectroscopy using a fiducial system for rapid, precise alignment and registration of the crystal positions is presented. This delivers unprecedented performance in serial crystallography experiments at room temperature under atmospheric pressure, giving a raw hit rate approaching 100% with an effective indexing rate of approximately 50%, increasing the efficiency of beam usage and allowing the method to be applied to systems where the number of crystals is limited.