Autoxidative and cyclooxygenase-2 catalyzed transformation of the dietary chemopreventive agent curcumin

The efficacy of the diphenol curcumin as a cancer chemopreventive agent is limited by its chemical and metabolic instability. Non-enzymatic degradation has been described to yield vanillin, ferulic acid, and feruloylmethane through cleavage of the heptadienone chain connecting the phenolic rings. Here we provide evidence for an alternative mechanism, resulting in autoxidative cyclization of the heptadienone moiety as a major pathway of degradation. Autoxidative transformation of curcumin was pH-dependent with the highest rate at pH 8 (2.2 μM/min) and associated with stoichiometric uptake of O(2). Oxidation was also catalyzed by recombinant cyclooxygenase-2 (COX-2) (50 nm; 7.5 μM/min), and the rate was increased ≈10-fold by the addition of 300 μM H(2)O(2). The COX-2 catalyzed transformation was inhibited by acetaminophen but not indomethacin, suggesting catalysis occurred by the peroxidase activity. We propose a mechanism of enzymatic or autoxidative hydrogen abstraction from a phenolic hydroxyl to give a quinone methide and a delocalized radical in the heptadienone chain that undergoes 5-exo cyclization and oxygenation. Hydration of the quinone methide (measured by the incorporation of O-18 from H(2)(18)O) and rearrangement under loss of water gives the final dioxygenated bicyclopentadione product. When curcumin was added to RAW264.7 cells, the bicyclopentadione was increased 1.8-fold in cells activated by LPS; vanillin and other putative cleavage products were negligible. Oxidation to a reactive quinone methide is the mechanistic basis of many phenolic anti-cancer drugs. It is possible, therefore, that oxidative transformation of curcumin, a prominent but previously unrecognized reaction, contributes to its cancer chemopreventive activity.     

The Cytoskeleton As A Target In Quinone Toxicity

The exposure of mammalian cells to toxic concentrations of redox cycling and alkylating quinones causes marked changes in cell surface structure knoun as plasma membrane blebbing. These alterations are associated with the redistribution of plasma membrane proteins and the disruption of the normal organization of the cytoskeletal microfilaments which appears 10 be due mainly to actin cross-linking and dissociation of α-actinin from the actin network. The major hiochemical mechanisms responsible for these effects seem to involve the depletion of cytoskeletal protein dfhydryl groups and the increase cytosolic Ca²⁺ concentration following the alkylation/oxidation of free sulfhydryl groups in several Ca²⁺ transport systems. Depletion of intracellular ATP is also associated with quinone-induced plasma menibrane blebbing. However, ATP depletion occurs well after the onset of the morphological changes. and thus it does not seem to be causatively related to their appeardncc Thiol reductants. such as dithiothreitol. efficiently prevent the oxidation of cytoskeletal protein thiols. the increase in cytosolic free Ca²⁺ concentration and cell blebbing induced by redox cycling. but not alkylating. quinones. These results demonstrate that alkylating and redox cycling quinones cause siinilar structural and biochemical modifications of the cytoskeleton by means of different mechanisms. namely alkylation and oxidation of critical sulfhydryl groups     

Design and synthesis of benzofuranic derivatives as new ligands at the melatonin-binding site MT3

Benzofuranic analogues of MCA-NAT (5-methoxycarbonylamino-N-acetyltryptamine) have been synthesized and evaluated as melatonin receptor ligands. Introduction of a methoxycarbonylamino substituent in the C-5 position of the benzofurane nucleus obtains MT₃ selective ligands. This selectivity can be modulated with suitable variations of the C-5 position and the acyl group on the C-3 side chain.     

Copper-containing amine oxidases. Biogenesis and catalysis; a structural perspective

This review will focus on how X-ray crystallographic studies of copper-containing amine oxidases have complemented the solution, kinetic, and spectroscopic research on this ubiquitous class of enzymes. These enzymes not only contain a copper ion at the active site, but also a unique organic cofactor, 2,4,5-trihydroxyphenylalanine quinone (TPQ), which is absolutely required for catalysis. Structural data have not only shed light on the catalytic mechanism of the enzyme, which converts primary amines, using molecular oxygen, to aldehydes, ammonia, and hydrogen peroxide, but also on biogenesis of the cofactor. The cofactor is derived from a tyrosine in the enzyme amino acid sequence and requires only the addition of copper(II) and molecular oxygen in a self-processing event.     

Electron transfer in quinoproteins

Soluble quinoprotein dehydrogenases oxidize a wide range of sugar, alcohol, amine, and aldehyde substrates. The physiological electron acceptors for these enzymes are not pyridine nucleotides but are other soluble redox proteins. This makes these enzymes and their electron acceptors excellent systems with which to study mechanisms of long-range interprotein electron transfer reactions. The tryptophan tryptophylquinone (TTQ)-dependent methylamine dehydrogenase (MADH) transfers electrons to a blue copper protein, amicyanin. It has been possible to alter the rate of electron transfer by using different redox forms of MADH, varying reaction conditions, and performing site-directed mutagenesis on these proteins. From kinetic and thermodynamic analyses of the reaction rates, it was possible to determine whether a change in rate is due a change in Delta G(0), electronic coupling, reorganization energy or kinetic mechanism. Examples of each of these cases are discussed in the context of the known crystal structures of the electron transfer protein complexes. The pyrroloquinoline quinone (PQQ)-dependent methanol dehydrogenase transfers electrons to a c-type cytochrome. Kinetic and thermodynamic analyses of this reaction indicated that this electron transfer reaction was conformationally coupled. Quinohemoproteins possess a quinone cofactor as well as one or more c-type hemes within the same protein. The structures of a PQQ-dependent quinohemoprotein alcohol dehydrogenase and a TTQ-dependent quinohemoprotein amine dehydrogenase are described with respect to their roles in intramolecular and intermolecular protein electron transfer reactions.     

Autoxidation of extracellular hydroquinones is a causative event for the cytotoxicity of menadione and DMNQ in A549-S cells

Cytotoxicity of 1,4-naphthoquinones has been attributed to intracellular reactive oxygen species (ROS) generation through one-electron-reductase-mediated redox cycling and to arylation of cellular nucleophiles. Here, however, we report that in a subclone of lung epithelial A549 cells (A549-S previously called A549-G4S (Watanabe, et al., Am. J. Physiol. 283 (2002) L726-736), the mechanism of ROS generation by menadione and by 2,3-dimethoxy-1,4-naphthoquinone (DMNQ), and therefore that of cytotoxicity, differs from the paradigm. Ninety percent of H(2)O(2) generation by both the quinones can be prevented by dicumarol, an inhibitor of NAD(P)H quinone oxidoreductase (NQO1), at the submicromolar level, regardless of the quinone concentrations. Exogenous SOD also inhibits H(2)O(2) production at low but not high concentrations of the quinones, especially DMNQ. Thus, at low quinone concentrations, superoxide-driven hydroquinone autoxidation accounts for more than half of H(2)O(2) generation by both quinones, whereas at high quinone concentrations, especially for DMNQ, comproportionation-driven hydroquinone autoxidation becomes the predominant mechanism. Hydroquinone autoxidation appears to occur predominantly in the extracellular environment than in the cytosol as extracellular catalase can dramatically attenuate quinone-induced cytotoxicity throughout the range of quinone concentrations, whereas complete inactivation of endogenous catalase or complete depletion of intracellular glutathione has only a marginal effect on their cytotoxicity. Finally, we show evidence that ROS production is a consequence of the compensatory defensive role of NQO1 against quinone arylation.     

Variation in proton donor/acceptor pathways in succinate:quinone oxidoreductases

The anaerobically expressed fumarate reductase and aerobically expressed succinate dehydrogenase from Escherichia coli comprise two different classes of succinate:quinone oxidoreductases (SQR), often termed respiratory complex II. The X-ray structures of both membrane-bound complexes have revealed that while the catalytic/soluble domains are structurally similar the quinone binding domains of the enzyme complexes are significantly different. These results suggest that the anaerobic and aerobic forms of complex II have evolved different mechanisms for electron and proton transfer in their respective membrane domains.     

Isolation and absolute stereochemistry of coussaric acid,a new bioactive triterpenoid from the stems of Coussarea brevicaulis

Coussaric acid (1), a triterpenoid based on an ursane skeleton, and an oleanane-type triterpene acid, 3-epi-spathodic acid (2), as well as four known compounds, barbinervic acid, scutellaric acid, stigmasterol and stigmasterol glucoside, have been isolated from an EtOAc-soluble extract of the stems of Coussarea brevicaulis. The structures of compounds 1 and 2 were elucidated on the basis of spectroscopic investigation, and single-crystal X-ray crystallography was used to confirm the structure of 1. The absolute stereochemistry of 1 was established by chemical transformations and by the Mosher ester procedure. The potential of the isolates and chemical transformation products to induce quinone reductase was evaluated in mouse Hepa lclc7 hepatoma cells.     

Quinone reductase 2 substrate specificity and inhibition pharmacology

Quinone reductase 2 is a mammalian cytosolic FAD-dependent enzyme, the activity of which is not supported by conventional nicotinamide nucleotides. An endobiotic substrate has never been reported for this enzyme nor a set of molecular tools, such as inhibitors. In the present work, we used the recombinant human enzyme, expressed in CHO cells for the systematic screening of both co-substrates and substrates. The co-substrates survey showed that the natural occurring compound, N-ribosylnicotinamide, was a poor co-substrate. The synthetic N-benzylnicotinamide is a better one compared to any other compounds tested. We found that tetrahydrofolic acid acted as a co-substrate for the reduction of menadione catalysed by quinone reductase 2, although with poor potency (Km approximately 2 mM). Among a series of commercially available quinones, a single one was found to be substrate of quinone reductase 2, in the presence of N-benzyldihydronicotinamide: coenzyme Q0. Finally, we tested a series of 197 flavonoids as potential inhibitors. We found apigenin, genistein or kaempferol as good inhibitor of quinone reductase 2 activity with IC50 in the 100 nM range. These compounds, co-substrate, substrate and inhibitors will permit to better know this enzyme, the role of which is still poorly understood.     

Salinivibrio costicola subsp. alcaliphilus subsp. nov., a haloalkaliphilic aerobe from Campania Region (Italy)

Phenotypic and phylogenetic studies were performed on unidentified Gram-negative staining, haloalkaliphilic aerobe and protease producer Salinivibrio-like organism recovered from a saltish spring with algal mat in the “Pozzo del Sale” site (Salt's Well) in the Campania Region (South Italy). Phylogenetic analysis based on comparison of 16S rRNA gene sequences demonstrated that the isolate was related to species of Salinivibrio genus. The DNA–DNA hybridization of the type strain 18AG^T with the most related Salinivibrio costicola subsp. costicola showed a re-association value of 72%. Based on the phenotypic distinctiveness of 18AG^T strain and molecular, chemical and genetic evidence, it is proposed that strain 18AG^T can be classified as S. costicola subsp. alcaliphilus, subsp. nov. The type strain of S. costicola subsp. alcaliphilus, is ATCC BAA-952^T; DSM 16359^T.