Getting the adrenaline going: Crystal structure of the adrenaline-synthesizing enzyme PNMT

Background: Adrenaline is localized to specific regions of the central nervous system (CNS), but its role therein is unclear because of a lack of suitable pharmacologic agents. Ideally, a chemical is required that crosses the blood-brain barrier, potently inhibits the adrenaline-synthesizing enzyme PNMT, and does not affect other catecholamine processes. Currently available PNMT inhibitors do not meet these criteria. We aim to produce potent, selective, and CNS-active PNMT inhibitors by structure-based design methods. The first step is the structure determination of PNMT. Results: We have solved the crystal structure of human PNMT complexed with a cofactor product and a submicromolar inhibitor at a resolution of 2.4 Angstrom. The structure reveals a highly decorated methyltransferase fold, with an active site protected from solvent by an extensive cover formed from several discrete structural motifs. The structure of PNMT shows that the inhibitor interacts with the enzyme in a different mode from the (modeled) substrate noradrenaline. Specifically, the position and orientation of the amines is not equivalent. Conclusions: An unexpected finding is that the structure of PNMT provides independent evidence of both backward evolution and fold recruitment in the evolution of a complex enzyme from a simple fold. The proposed evolutionary pathway implies that adrenaline, the product of PNMT catalysis, is a relative newcomer in the catecholamine family. The PNMT structure reported here enables the design of potent and selective inhibitors with which to characterize the role of adrenaline in the CNS. Such chemical probes could potentially be useful as novel therapeutics.

Crystal structure of yeast acetohydroxyacid synthase: A target for herbicidal inhibitors

Acetohydroxyacid synthase (AHAS; EC 4.1.3.18) catalyzes the first step in branched-chain amino acid biosynthesis. The enzyme requires thiamin diphosphate and FAD for activity, but the latter is unexpected, because the reaction involves no oxidation or reduction. Due to its presence in plants, AHAS is a target for sulfonylurea and imidazolinone herbicides. Here, the crystal structure to 2.6 A resolution of the catalytic subunit of yeast AHAS is reported. The active site is located at the dimer interface and is near the proposed herbicide-binding site. The conformation of FAD and its position in the active site are defined. The structure of AHAS provides a starting point for the rational design of new herbicides. (C) 2002 Elsevier Science Ltd.

Crystal structures of free, IMP-, and GMP-bound Escherichia coli hypoxanthine phosphoribosyltransferase

Crystal structures have been determined for free Escherichia coli hypoxanthine phosphoribosyltransferase (HPRT) (2.9 Angstrom resolution) and for the enzyme in complex with the reaction products, inosine 5'-monophosphate (IMP) and guanosine 5-monophosphate (GMP) (2.8 Angstrom resolution). Of the known 6-oxopurine phosphoribosyltransferase (PRTase) structures, E. coli HPRT is most similar in structure to that of Tritrichomonas foetus HGXPRT, with a rmsd for 150 Calpha atoms of 1.0 Angstrom. Comparison of the free and product bound structures shows that the side chain of Phe156 and the polypeptide backbone in this vicinity move to bind IMP or GMP. A nonproline cis peptide bond, also found in some other 6-oxopurine PRTases, is observed between Leu46 and Arg47 in both the free and complexed structures. For catalysis to occur, the 6-oxopurine PRTases have a requirement for divalent metal ion, Usually Mg2+ in vivo. In the free structure, a Mg2+, is coordinated to the side chains of Glu103 and Asp104. This interaction may be important for stabilization of the enzyme before catalysis. E. coli HPRT is unique among the known 6-oxopurine PRTases in that it exhibits a marked preference for hypoxanthine as substrate over both xanthine and guanine. The structures suggest that its substrate specificity is due to the modes of binding of the bases. In E. coli HPRT, the carbonyl oxygen of Asp 163 would likely form a hydrogen bond with the 2-exocyclic nitrogen of guanine (in the HPRT-guanine-PRib-PP-Mg2+ complex). However, hypoxanthine does not have a 2-exocyclic atom and the HPRT-IMP structure suggests that hypoxanthine is likely to occupy a different position in the purine-binding pocket.

Dynamic Behavior of the Jahn-Teller distorted Cu(H2O)(6)(2+) ion in Cu2+ doped Cs-2[Zn(H2O)(6)](ZrF6)(2) and the crystal structure of the host lattice

The temperature dependence of the X- and Q-band EPR spectra of Cs-2[Zn(H2O)(6)](ZrF6)(2) containing similar to1% Cu2+ is reported. All three molecular g-values vary with temperature, and their behavior is interpreted using a model in which the potential surface of the Jahn-Teller distorted Cu(H2O)(6)(2+) ion is perturbed by an orthorhombic strain induced by interactions with the surrounding lattice. The strain parameters are significantly smaller than those reported previously for the Cu(H2O)(6)(2+) ion in similar lattices. The temperature dependence of the two higher g-values suggests that in the present compound the lattice interactions change slightly with temperature. The crystal structure of the Cs-2[Zn(H2O)(6)](ZrF6)(2) host is reported, and the geometry of the Zn(H2O)(6)(2+) ion is correlated with lattice strain parameters derived from the EPR spectrum of the guest Cu2+ complex.

Synthesis and characterization of mono- and bis-ligand zinc(II) and cadmium(II) complexes of the di-2-pyridylketone Schiff base of S-benzyl dithiocarbazate (Hdpksbz) and the X-ray crystal structures of the [Zn(dpksbz)2] and [Cd(dpksbz)NCS]2 complexes

New mono- and bis-chelated zinc(II) and cadmium(II) complexes of formula, [M(dpksbz)NCS] (dpksbz = anionic form of the di-2-pyridylketone Schiff base of S-benzyldithiocarbazate) and [M(dpksbz)(2)] (M = Zn-II, Cd-II) have been prepared and characterized. The structure of the bis-ligand complex, [Zn(dpksbZ)(2)] has been determined by X-ray diffraction. The complex has a distorted octahedral geometry in which the ligands are coordinated to the zinc(II) ion as uninegatively charged tridentate chelates via the thiolate sulfur atoms, the azomethine nitrogen atoms and the pyridine nitrogen atoms. The distortion from a regular octahedral geometry is attributed to the restricted bite angles of the Schiff base ligands. X-ray structural analysis shows that the [Cd(dpksbz)NCS](2) complex is a centrosymmetric dimer in which each of the cadmium(II) ions adopts a five-coordinate, approximately square-pyramidal configuration with the Schiff base acting as a tetradentate chelating agent coordinating a cadmium(II) ion via one of the pyridine nitrogen atoms, the azomethine nitrogen atom and the thiolate sulfur atom; the second pyridine nitrogen atom is coordinated to the other cadmium(II) ion of the dimer. The fifth coordination position around each cadmium(II) is occupied by an N-bonded thiocyanate ligand. (C) 2003 Elsevier Science Ltd. All rights reserved.

The preparation of zinc(II) and cadmium(II) complexes of the pentadentate N3S2 ligand formed from 2,6-diacetylpyridine and S-benzyldithiocarbazate (H2SNNNS) and the X-ray crystal structure of the novel dimeric [Zn2(SNNNS)2] complex

The pentadentate chelating agent, 2,6-diacetylpyridinebis(S-benzyldithiocarbazate) (H2SNNNS) reacts with zinc(II) and cadmium(II) ions forming stable complexes of empirical formula, [M(SNNNS)] (M=Zn2+, Cd2+; SNNNS2 =doubly deprotonated anionic form of the Schiff base). These complexes have been characterized by a variety of physico-chemical techniques. IR and H-1 NMR spectral evidence indicate that the Schiff base coordinates to the zinc(II) and cadmium(II) ions via the pyridine nitrogen atoms, the azomethine nitrogen atoms and the mercaptide sulfur atoms. The crystal and molecular structure of the zinc(II) complex has been determined by X-ray diffraction. The complex is a dimer in which the pyridine nitrogen atom,the azomethine nitrogen atom and the thiolate sulfur atom from one ligand coordinate to one of the zinc(II) ions whereas the azomethine and thiolate sulfur atoms from another ligand complete pentacoordination around the zinc(II) ion, the ligands being coordinated in their deprotonated forms. The coordination geometry about each zinc(II) can be considered as intermediate between a square-pyramid and trigonal-bipyramid. The cadmium(II) complex is also assigned with a dimeric structure. (C) 2003 Elsevier Ltd. All rights reserved.

Low-temperature single-crystal Raman and neutron-diffraction study of the hydrogenous ammonium copper(II) tutton salt and the deuterated analogue in the metastable state

Low-temperature (15 K) single-crystal neutron-diffraction structures and Raman spectra of the salts (NX4)(2)[CU(OX2)(6)](SO4)(2), where X = H or D, are reported. This study is concerned with the origin of the structural phase change that is known to occur upon deuteration. Data for the deuterated salt were measured in the metastable state, achieved by application of 500 bar of hydrostatic pressure at similar to303 K followed by cooling to 281 K and the subsequent release of pressure. This allows for the direct comparison between the hydrogenous and deuterated salts, in the same modification, at ambient pressure and low temperature. The Raman spectra provide no intimation of any significant change in the intermolecular bonding. Furthermore, structural differences are few, the largest being for the long Cu-O bond, which is 2.2834(5) and 2.2802(4) Angstrom for the hydrogenous and the deuterated salts, respectively. Calorimetric data for the deuterated salt are also presented, providing an estimate of 0.17(2) kJ/mol for the enthalpy difference between the two structural forms at 295.8(5) K. The structural data suggest that substitution of hydrogen for deuterium gives rise to changes in the hydrogen-bonding interactions that result in a slightly reduced force field about the copper(II) center. The small structural differences suggest different relative stabilities for the hydrogenous and deuterated salts, which may be sufficient to stabilize the hydrogenous salt in the anomalous structural form.

Dehydration converts DsbG crystal diffraction from low to high resolution

Diffraction quality crystals are essential for crystallographic studies of protein structure, and the production of poorly diffracting crystals is often regarded as a dead end in the process. Here we show a dramatic improvement of poorly diffracting DsbG crystals allowing high-resolution diffraction data measurement. Before dehydration, the crystals are fragile and the diffraction pattern is streaky, extending to 10 Angstrom resolution. After dehydration, there is a spectacular improvement, with the diffraction pattern extending to 2 Angstrom resolution. This and other recent results show that dehydration is a simple, rapid, and inexpensive approach to convert poor quality crystals into diffraction quality crystals.

The synthesis and X-ray crystal structure of 6-bromo-2,4,4-trimethyl-cyclohex-2-enone

Regiospecific bromination of 2,4,4-trimethyl-cyclohex-2-enone was achieved and the X-ray crystal structure of 6-bromo-2,4,4-trimethyl-cyclohex-2-enone is presented.

The synthesis and X-ray crystal structure of 9-carboxyhexahydro-7-methoxy-4a,7-ethano-benzopyran-5-en-1-one

9-Carboxyhexahydro-7-methoxy-4a,7-ethano-benzopyran-5-en-1-one (1) was prepared and examined by X-ray crystallography to probe its potential as a new peptide scaffold/template. The crystal structure of the anhydride precursor 7-(2-acetoxyethyl)-4-methoxy-3a,4,7,7a-tetrahydro-4,7-ethanoisobenzofuran-1,3-dione (6) is also reported.