Summary Lipid biology continues to emerge as an area of significant therapeutic interest, particularly as the result of an enhanced understanding of the wealth of signaling molecules with diverse physiological properties. This growth in knowledge is epitomized by lysophosphatidic acid (LPA), which functions through interactions with six cognate G protein-coupled receptors. Herein we present three crystal structures of LPA1 in complex with antagonist tool compounds selected and designed through structural and stability analysis. Structural analysis combined with molecular dynamics identified a basis for ligand access to the LPA1 binding pocket from the extracellular space contrasting with the proposed access for the sphingosine 1-phosphate receptor. Characteristics of the LPA1 binding pocket raise the possibility of promiscuous ligand recognition of phosphorylated endocannabinoids. Cell-based assays confirmed this hypothesis, linking the distinct receptor systems through metabolically related ligands with potential functional and therapeutic implications for treatment of disease.
CTP synthetase (CTPs) catalyzes the last step in CTP biosynthesis, in which ammonia generated at the glutaminase domain reacts with the ATP-phosphorylated UTP at the synthetase domain to give CTP. Glutamine hydrolysis is active in the presence of ATP and UTP and is stimulated by the addition of GTP. We report the crystal structures of Thermus thermophilus HB8 CTPs alone, CTPs with 3SO4(2-), and CTPs with glutamine. The enzyme is folded into a homotetramer with a cross-shaped structure. Based on the binding mode of sulfate anions to the synthetase site, ATP and UTP are computer modeled into CTPs with a geometry favorable for the reaction. Glutamine bound to the glutaminase domain is situated next to the triad of Glu-His-Cys as a catalyst and a water molecule. Structural information provides an insight into the conformational changes associated with the binding of ATP and UTP and the formation of the GTP binding site.
Fluoroacetate dehalogenase catalyzes the hydrolytic defluorination of fluoroacetate to produce glycolate. The enzyme is unique in that it catalyzes the cleavage of a carbon-fluorine bond of an aliphatic compound: the bond energy of the carbon-fluorine bond is among the highest found in natural products. The enzyme also acts on chloroacetate, although much less efficiently. We here determined the X-ray crystal structure of the enzyme from Burkholderia sp. strain FA1 as the first experimentally determined three-dimensional structure of fluoroacetate dehalogenase. The enzyme belongs to the ␣/ hydrolase superfamily and exists as a homodimer. Each subunit consists of core and cap domains. The catalytic triad, Asp104-His271-Asp128, of which Asp104 serves as the catalytic nucleophile, was found in the core domain at the domain interface. The active site was composed of Phe34, Asp104, Arg105, Arg108, Asp128, His271, and Phe272 of the core domain and Tyr147, His149, Trp150, and Tyr212 of the cap domain. An electron density peak corresponding to a chloride ion was found in the vicinity of the N 1 atom of Trp150 and the N 2 atom of His149, suggesting that these are the halide ion acceptors. Site-directed replacement of each of the active-site residues, except for Trp150, by Ala caused the total loss of the activity toward fluoroacetate and chloroacetate, whereas the replacement of Trp150 caused the loss of the activity only toward fluoroacetate. An interaction between Trp150 and the fluorine atom is probably an absolute requirement for the reduction of the activation energy for the cleavage of the carbon-fluorine bond.Fluoroacetate is a naturally occurring organofluorine compound. An actinomycete, "Streptomyces cattleya" (30), and some plants in Australia and Africa (2) produce this highly toxic compound. Fluoroacetate has a carbon-fluorine bond, whose dissociation energy is among the highest found in nature (8). Despite this fact, fluoroacetate dehalogenases (FAcDEXs) from Burkholderia sp. strain FA1 (FAc-DEX FA1) (20) and Delftia acidovorans strain B (formerly Moraxella sp. strain B; FAc-DEX H1) (16) catalyze the hydrolytic defluorination of fluoroacetate to produce glycolate. These enzymes are unique in that they catalyze the cleavage of the carbon-fluorine bond, which is much stronger than other carbon-halogen bonds.FAc-DEXs show weak but significant sequence similarity to proteins that belong to the ␣/ hydrolase superfamily, such as epoxide hydrolases from Agrobacterium radiobacter AD1 (37) and humans and haloalkane dehalogenases from Sphingobium japonicum UT26 (formerly Sphingomonas paucimobilis UT26) (32) and Xanthobacter autotrophicus GJ10 (17). Although haloalkane dehalogenases of this superfamily catalyze the hydrolytic cleavage of carbon-halogen bonds, none of them catalyzes the cleavage of a carbon-fluorine bond. Dehalogenases that belong to other families, such as L-2-haloacid dehalogenase (22), DL-2-haloacid dehalogenase (28), and haloalcohol dehalogenase (42), also do not catalyze the cleavage of a carbon-...
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