Oxalate decarboxylase (EC 4.1.1.2) catalyzes the conversion of oxalate to formate and carbon dioxide and utilizes dioxygen as a cofactor. By contrast, the evolutionarily related oxalate oxidase (EC 1.2.3.4) converts oxalate and dioxygen to carbon dioxide and hydrogen peroxide. Divergent free radical catalytic mechanisms have been proposed for these enzymes that involve the requirement of an active site proton donor in the decarboxylase but not the oxidase reaction. The oxidase possesses only one domain and manganese binding site per subunit, while the decarboxylase has two domains and two manganese sites per subunit. A structure of the decarboxylase together with a limited mutagenesis study has recently been interpreted as evidence that the C-terminal domain manganese binding site (site 2) is the catalytic site and that Glu-333 is the crucial proton donor (Anand, R., Dorrestein, P. C., Kinsland, C., Begley, T. P., and Ealick, S. E. (2002) Biochemistry 41, 7659 -7669).The N-terminal binding site (site 1) of this structure is solvent-exposed (open) and lacks a suitable proton donor for the decarboxylase reaction. We report a new structure of the decarboxylase that shows a loop containing a 3 10 helix near site 1 in an alternative conformation. This loop adopts a "closed" conformation forming a lid covering the entrance to site 1. This conformational change brings Glu-162 close to the manganese ion, making it a new candidate for the crucial proton donor. Sitedirected mutagenesis of equivalent residues in each domain provides evidence that Glu-162 performs this vital role and that the N-terminal domain is either the sole or the dominant catalytically active domain.
Bacillus subtilis has been shown to express a cytosolic oxalate decarboxylase (EC 4.1.1.2 ). The enzyme was induced in acidic growth media, particularly at pH 5.0, but not by oxalate. The enzyme was purified, and N-terminal sequencing identified the protein to be encoded by yvrK. The role of the first oxalate decarboxylase to be identified in a prokaryote is discussed.
Bloom's syndrome is an autosomal recessive genome-instability disorder associated with a predisposition to cancer, premature aging and developmental abnormalities. It is caused by mutations that inactivate the DNA helicase activity of the BLM protein or nullify protein expression. The BLM helicase has been implicated in the alternative lengthening of telomeres (ALT) pathway, which is essential for the limitless replication of some cancer cells. This pathway is used by 10-15% of cancers, where inhibitors of BLM are expected to facilitate telomere shortening, leading to apoptosis or senescence. Here, the crystal structure of the human BLM helicase in complex with ADP and a 3'-overhang DNA duplex is reported. In addition to the helicase core, the BLM construct used for crystallization (residues 640-1298) includes the RecQ C-terminal (RQC) and the helicase and ribonuclease D C-terminal (HRDC) domains. Analysis of the structure provides detailed information on the interactions of the protein with DNA and helps to explain the mechanism coupling ATP hydrolysis and DNA unwinding. In addition, mapping of the missense mutations onto the structure provides insights into the molecular basis of Bloom's syndrome.
YvrK was found to contain between 0.86 and 1.14 atoms of manganese/subunit. EPR spectroscopy showed that the metal ion was predominantly but not exclusively in the Mn(II) oxidation state. The hyperfine coupling constant (A ؍ 9.5 millitesla) of the main g ؍ 2 signal was consistent with oxygen and nitrogen ligands with hexacoordinate geometry. The structure of YvrK was modeled on the basis of homology with oxalate oxidase, canavalin, and phaseolin, and its hexameric oligomerization was predicted by analogy with proglycinin and homogentisate 1,2-dioxygenase. Although YvrK possesses two potential active sites, only one could be fully occupied by manganese. The possibility that the C-terminal domain active site has no manganese bound and is buried in an intersubunit interface within the hexameric enzyme is discussed. A mechanism for oxalate decarboxylation is proposed, in which both Mn(II) and O 2 are cofactors that act together as a two-electron sink during catalysis.
Interleukin-2 tyrosine kinase, Itk, is an important member of the Tec family of non-receptor tyrosine kinases that play a central role in signaling through antigen receptors such as the T-cell receptor, B-cell receptor, and Fc⑀. Selective inhibition of Itk may be an important way of modulating many diseases involving heightened or inappropriate activation of the immune system. In addition to an unliganded nonphophorylated Itk catalytic kinase domain, we determined the crystal structures of the phosphorylated and nonphosphorylated kinase domain bound to staurosporine, a potent broad-spectrum kinase inhibitor. These structures are useful for the design of novel, highly potent and selective Itk inhibitors and provide insight into the influence of inhibitor binding and phosphorylation on the conformation of Itk.The Tec kinases are a family of five non-receptor tyrosine kinases that play a central role in signaling through antigenreceptors such as the T-cell receptor (TCR), 1 B-cell receptor, and Fc⑀ (1) and are essential for T-cell activation. Three members of the family, Itk, Rlk, and Tec, are activated downstream of antigen receptor engagement in T-cells and transmit signals to downstream effectors, including PLC-␥. A fourth member, Btk, appears to act independently of T-cell signaling and is essential for B-cell development and activation. Btk-deficient murine mast cells have reduced degranulation and decreased production of proinflammatory cytokines following Fc⑀RI crosslinking (2). Btk deletion in mice has a profound effect on B-cell proliferation induced by anti-IgM and inhibits immune responses to thymus-independent type II antigens (3, 4). A biological role for the final member of this family, Bmx, has not been identified.Itk is a key member of this family, and a number of factors point to the importance of this kinase in immune disease.Deletion of Itk in mice results in reduced TCR-induced proliferation and secretion of the cytokines IL-2, IL-4, IL-5, IL-10, and interferon-␥ (5-7). Also, the immunological symptoms of allergic asthma are attenuated in ItkϪ/Ϫ mice, and lung inflammation, eosinophil infiltration, and mucous production are drastically reduced in response to challenge with the allergen OVA (8). Furthermore, the Itk gene is reported to be more highly expressed in peripheral blood T-cells from patients with moderate or severe atopic dermatitis than in controls or patients with mild atopic dermatitis (9).In certain cell types, the role of Itk may be intricately linked with other members of the family. For example, in mast cells, Btk and Itk are both expressed and activated by Fc⑀RI crosslinking (10). Splenocytes from RlkϪ/Ϫ mice secrete half the IL-2 produced by wild type animals in response to TCR engagement (5), whereas the combined deletion of Itk and Rlk in mice leads to a profound inhibition of TCR-induced responses, including proliferation and production of the cytokines IL-2, IL-4, IL-5, and interferon-␥ (5, 7). Furthermore, intracellular signaling following TCR engagement is affected in Itk...
Protein kinase C θ (PKCθ) has a central role in T cell activation and survival; however, the dependency of T cell responses to the inhibition of this enzyme appears to be dictated by the nature of the antigen and by the inflammatory environment. Studies in PKCθ-deficient mice have demonstrated that while antiviral responses are PKCθ-independent, T cell responses associated with autoimmune diseases are PKCθ-dependent. Thus, potent and selective inhibition of PKCθ is expected to block autoimmune T cell responses without compromising antiviral immunity. Herein, we describe the development of potent and selective PKCθ inhibitors, which show exceptional potency in cells and in vivo. By use of a structure based rational design approach, a 1000-fold improvement in potency and 76-fold improvement in selectivity over closely related PKC isoforms such as PKCδ were obtained from the initial HTS hit, together with a big improvement in lipophilic efficiency (LiPE).
Interleukin-2 inducible T-cell kinase (Itk) plays a role in T-cell functions, and its inhibition potentially represents an attractive intervention point to treat autoimmune and allergic diseases. Herein we describe the discovery of a series of potent and selective novel inhibitors of Itk. These inhibitors were identified by structure-based design, starting from a fragment generated de novo, the 3-aminopyrid-2-one motif. Functionalization of the 3-amino group enabled rapid enhancement of the inhibitory activity against Itk, while introduction of a substituted heteroaromatic ring in position 5 of the pyridone fragment was key to achieving optimal selectivity over related kinases. A careful analysis of the hydration patterns in the kinase active site was necessary to fully explain the observed selectivity profile. The best molecule prepared in this optimization campaign, 7v, inhibits Itk with a K(i) of 7 nM and has a good selectivity profile across kinases.
An arylketone monooxygenase was purified from Pseudomonas putida JD1 by ion exchange and affinity chromatography. It had the characteristics of a Baeyer-Villiger-type monooxygenase and converted its substrate, 4-hydroxyacetophenone, into 4-hydroxyphenyl acetate with the consumption of one molecule of oxygen and oxidation of one molecule of NADPH per molecule of substrate. The enzyme was a monomer with an M r of about 70,000 and contained one molecule of flavin adenine dinucleotide (FAD). The enzyme was specific for NADPH as the electron donor, and spectral studies showed rapid reduction of the FAD by NADPH but not by NADH. Other arylketones were substrates, including acetophenone and 4-hydroxypropiophenone, which were converted into phenyl acetate and 4-hydroxyphenyl propionate, respectively. The enzyme displayed MichaelisMenten kinetics with apparent K m values of 47 M for 4-hydroxyacetophenone, 384 M for acetophenone, and 23 M for 4-hydroxypropiophenone. The apparent K m value for NADPH with 4-hydroxyacetophenone as substrate was 17.5 M. The N-terminal sequence did not show any similarity to other proteins, but an internal sequence was very similar to part of the proposed NADPH binding site in the Baeyer-Villiger monooxygenase cyclohexanone monooxygenase from an Acinetobacter sp.One of the strategies used by microorganisms for the aerobic metabolism of ketones in degradative processes is to perform the biological equivalent of the Baeyer-Villiger reaction. This involves the insertion of an oxygen between the keto carbon and an adjacent carbon to form an ester, which can then be readily hydrolyzed. This sequence has been described for the degradation of a number of cyclic ketones, for example cyclohexanone and camphor, which yield lactones on oxygenation (6,20), and for the splitting of aliphatic ketones such as 2-tridecanone (1) or the removal of the side chain of progesterone (13). Several of the enzymes involved in the oxygenating reaction have been purified, and the cyclohexanone monooxygenase from an Acinetobacter species has been studied in great detail and particularly well characterized (2, 7, 21). Generally they have the properties of flavoprotein monooxygenases with a requirement for reduced NAD or NADP, although the enzymes that lactonize the enantiomers of camphor require an additional protein to transfer electrons from the NADH to the monooxygenase component (20). Some of these enzymes have broad substrate specificities, including the ability to oxidize sulfur or selenium atoms in organic compounds, and they are capable of forming chiral products in high enantiomeric excess. Consequently, there has been considerable interest in this class of enzyme because of the potential use for the biological production of chiral synthons, and some of these enzymes are now produced commercially (14,15).In addition to the metabolism of cyclic and aliphatic ketones, the degradation of aryl ketones can also proceed by formation of an ester, apparently by the action of a Baeyer-Villiger type of monooxygenase. In the...
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