Biomimetic polyene cyclizations: A review

It has been shown that certain polyenic substances, having trans olefinic bonds in the 1,5 relationship, can be induced to undergo stereospecific, nonenzymic, cationic cyclization to give polycyclic products with the all trans (“natural”) configuration. These transformations appear to mimic in principle the biogenetic conversion of squalene into polycyclic triterpenoids, e.g., lanosterol. Acetal as well as allylic alcohol functions have proved to be effective initiators for such cyclizations, many of which proceed to a remarkable degree of completion giving mainly totally cyclized products. Thus, it has been possible to convert, in a single step, an open chain tetraenic acetal having no chiral centers, into a tetracyclic product having seven such centers. The process is highly stereoselective giving only two of 64 possible racemates.Methylacetylenic and also styryl end groups are particularly useful cyclization terminators as they provide a means of realizing five-membered ring formation. Systems with these terminators have been developed for effecting the total synthesis of the steroid nucleus in a single step starting from a substrate containing only one ring.The mechanism of these biomimetic as well as of the enzymic cyclizations is open to question, but the balance of the evidence is somewhat in favor of a synchronous process.     

Anaerobic Metabolism of Cyclohex-1-Ene-1-Carboxylate, a Proposed Intermediate of Benzoate Degradation, by Rhodopseudomonas palustris

Anaerobic benzoate degradation by the phototrophic bacterium Rhodopseudomonas palustris has been proposed to proceed via aromatic ring reduction reactions leading to cyclohex-1-ene-1-carboxyl-coenzyme A (CoA) formation. The alicyclic product is then proposed to undergo three β-oxidation-like modifications resulting in ring cleavage. Illuminated suspensions of benzoate-grown cells converted [7-¹⁴C]cyclohex-1-ene-1-carboxylate to intermediates that comigrated with cyclohex-1-ene-1-carboxyl-CoA, 2-hydroxycyclohexanecar-boxyl-CoA, 2-ketocyclohexanecarboxyl-CoA, and pimelyl-CoA by thin-layer chromatography. This set of intermediates was also formed by cells grown anaerobically or aerobically on cyclohex-1-ene-1-carboxylate, indicating that benzoate-grown and cyclohex-1-ene-1-carboxylate-grown cells degrade this alicyclic acid by the same catabolic route. Four enzymatic activities proposed to be required for conversion of cyclohex-1-ene-1-carboxylate to pimelyl-CoA were detected at 3- to 10-fold-higher levels in benzoate-grown cells than in succinate-grown cells. These were cyclohex-1-ene-1-carboxylate-CoA ligase, cyclohex-1-ene-1-carboxyl-CoA hydratase, 2-hydroxycyclohexanecarboxyl-CoA dehydrogenase, and 2-ketocyclohexanecarboxyl-CoA hydrolase (ring cleaving). Pimelyl-CoA was identified in hydrolase reaction mixtures as the product of alicyclic ring cleavage. The results provide a first demonstration of an alicyclic ring cleavage activity.     

Membrane potential difference of isolated plant vacuoles: positive or negative? I. Evidence for membrane binding of cationic probes

The binding of membrane potential cationic probes was studied on phospholipidic liposomes by equilibrium dialysis and microelectrophoresis. Surface binding of lipophilic cations (benzyltributylammonium or tetraphenylphosphonium) appears to be the major accumulation mechanism in liposomes and simulates the existence of a negative transmembrane potential (E_m), in absence of any transmembrane ionic gradient. Furthermore, this apparent negative potential has a classical response with regard to common E_m effectors, namely a depolarization induced by KCl or FCCP (carbonylcyanide p-trifluoromethoxyphenylhydrazone). The relevance of these results to the study of transtonoplast potential difference on isolated vacuoles was investigated. Tetraphenylphosphonium was shown to bind to the tonoplast, the essential features of binding and interaction with E_m effectors being similar in vacuoles and liposomes. Therefore the assumption of negligible binding of cationic probe to vacuoles, classically admitted in determinations of vacuolar E_m using lipophilic cations, is untenable.     

Fluorinated non-imidazole histamine H3 receptor antagonists

Fluorine substituents have become a widespread and important component in drug design and development. Here, the synthesis of fluorine containing compounds and some corresponding precursor molecules are presented for potential isotope labelling as well as their data obtained with in vitro and in vivo screenings. The compounds vary in the basic centres (piperidine or pyrrolidine) and are fluoro substituted in different positions of the basic alicyclic moiety. Pharmacological evaluation resulted in ligands with high affinities at hH₃ receptor in the nanomolar and subnanomolar concentration range and some with high antagonist in vivo potencies.     

Monoterpene cyclases. Stereoelectronic requirements for substrate binding and ionization

Enzymes from Salvia officinalis, capable of catalyzing the electrophilic isomerization and subsequent cyclization of geranyl pyrophosphate (3,8-dimethylocta-2E,6-dienyl pyrophosphate) to the monoterpenes (+)-alpha-pinene and (+)-bornyl pyrophosphate, were examined with a series of substrate analogs modified in carbon chain length and in the geometric and electronic character of the C2-C3 and C6-C7 olefinic domains. Inhibition studies with these monoterpene cyclases indicated that the pyrophosphate ester function was the principal determinant of substrate recognition and that the C2-C3 olefin was recognized largely on the basis of geometry, whereas the primary basis of interaction with the C6-C7 olefin was electronic. A related group of allylic pyrophosphates was tested for the ability to undergo enzyme-catalyzed ionization to afford olefinic and/or alcoholic products. From the relative reaction rates it was deduced that the alignment of the allylic pi-system with the C1-OP bond was essential for ionization of the substrate and that specific interaction with the distal C6-C7 isopropylidene function served not only to optimize orbital alignment but also to exclude water from the active site, and thus determine the partitioning of cationic intermediates into olefins or alcohols. From the combination of results, the interrelationships of substrate functional groups within the active site could be approximated and the topology of geranyl pyrophosphate binding to the cyclase thereby formulated.     

Transnitrosation of alicyclic N-nitrosamines containing a sulfur atom

Aromatic and aliphatic nitrosamines are known to transfer a nitrosonium ion to another amine. The transnitrosation of alicyclic N-nitroso compounds generates S-nitrosothiols, which are potential nitric oxide donors in vivo. In this study, certain alicyclic N-nitroso compounds based on non-mutagenic N-nitrosoproline or N-nitrosothioproline were synthesised, and the formation of S-nitrosoglutathione (GSNO) was quantified under acidic conditions. We then investigated the effect of a sulfur atom as the substituent and as a ring component on the GSNO formation. In the presence of thiourea under acidic conditions, GSNO was formed from N-nitrosoproline and glutathione, and an N-nitroso compound containing a sulfur atom and glutathione produced GSNO without thiourea. The quantity of GSNO derived from the reaction of the N-nitrosamines containing a sulfur atom and glutathione was higher than that from the N-nitrosoproline and glutathione plus thiourea. Among the analogues that contained a sulfur atom either in the ring or as a substituent, the thiazolidines produced a slightly higher quantity of GSNO than the analogue with a thioamide group. A compound containing sulfur atoms both in the ring and as a substituent exhibited the highest activity for GSNO formation among the alicyclic N-nitrosamines tested. The results indicate that the intramolecular sulfur atom plays an important role in the transnitrosation via alicyclic N-nitroso compounds to form GSNO.     

DNA-Induced Aggregation and Fusion of Phosphatidylcholine Liposomes in the Presence of Multivalent Cations Observed by the Cryo-TEM Technique

By means of cryoelectron transmission microscopy (cryo-TEM), we were able to demonstrate the formation of ternary complexes (TC): DNA–phosphatidylcholine liposome–divalent metal cations. Addition of Ba²⁺ to TC led to visualization of DNA compacting on the liposome surface. Staining the TC by Tb³⁺ cations revealed the changed secondary structure of DNA located between fused liposomes. Cryo-TEM and liposome turbidity data were analyzed during TC formation. Liposome aggregation and the liposome fusion induced by DNA in TC were observed. Because TC displayed the property of DNA cationic liposome complexes as well as their own unique properties, we were able to consider cationic lipoplexes as a particular case of TC. The involvement of TC and direct DNA–lipid interactions in the formation nuclear pore complexes were assumed.     

Biodegradation of an Alicyclic Hydrocarbon by a Sulfate-Reducing Enrichment from a Gas Condensate-Contaminated Aquifer

We used ethylcyclopentane (ECP) as a model alicyclic hydrocarbon and investigated its metabolism by a sulfate-reducing bacterial enrichment obtained from a gas condensate-contaminated aquifer. The enrichment coupled the consumption of ECP with the stoichiometrically expected amount of sulfate reduced. During ECP biodegradation, we observed the transient accumulation of metabolite peaks by gas chromatography-mass spectrometry, three of which had identical mass spectrometry profiles. Mass-spectral similarities to analogous authentic standards allowed us to identify these metabolites as ethylcyclopentylsuccinic acids, ethylcyclopentylpropionic acid, ethylcyclopentylcarboxylic acid, and ethylsuccinic acid. Based on these findings, we propose a pathway for the degradation of this alicyclic hydrocarbon. Furthermore, a putative metabolite similar to ethylcyclopentylsuccinic acid was also found in samples of contaminated groundwater from the aquifer. However, no such finding was evident for samples collected from wells located upgradient of the gas condensate spill. Microbial community analysis of the ECP-degrading enrichment by denaturing gradient gel electrophoresis revealed the presence of at least three different organisms using universal eubacterial primers targeting 550 bp of the 16S rRNA gene. Based on sequence analysis, these organisms are phylogenetically related to the genera Syntrophobacter and Desulfotomaculum as well as a member of the Cytophaga-Flexibacter-Bacteroides group. The evidence suggests that alicyclic hydrocarbons such as ECP can be anaerobically activated by the addition to the double bond of fumarate to form alkylsuccinate derivatives under sulfate-reducing conditions and that the reaction occurs in the laboratory and in hydrocarbon-impacted environments.     

N-methylimidazolium chloride-catalyzed pyrophosphate formation: application to the synthesis of Lipid I and NDP-sugar donors

N-Methylimidazolium chloride is found to catalyze a coupling reaction between monophosphates and activated phosphorous-nitrogen intermediates such as a phosphorimidazolide and phosphoromorpholidate to form biologically important unsymmetrical pyrophosphate diesters. The catalyst is much more active, cheaper, and less explosive than 1H-tetrazole, known as the best catalyst for the pyrophosphate formation over a decade. The mild and neutral reaction conditions are compatible with allylic pyrophosphate formation in Lipid I syntheisis. ³¹P NMR experiments suggest that the catalyst acts not only as an acid but also as a nucleophile to form cationic and electrophilic phosphor-N-methylimidazolide intermediates in the pyrophosphate formation.     

Synthesis and antifungal activities of hydrophilic cationic polymers against Rhizoctonia solani

A series of linear hydrophilic cationic polymers with different charge density and molecular weights were synthesized by one-step polymerization process. The effect of the hydrophobicity and molecular weights on the antifungal activity against Rhizoctonia solani (R. solani) and Fusarium oxysporum f. sp. cubense race 4 (Foc4) was assessed. The biotoxicity of the cationic polymers were evaluated based on their median lethal concentration (LC₅₀) for zebrafish and silkworm and median lethal dose (LD₅₀) for Kunming mice. The results indicated that the balance between antifungal activity and biotoxicity could be well tuned by controlling the hydrophobic-hydrophilic balance. The minimum inhibitory concentration (MIC) of PEPB10 and PEPB25 against R. solani were 160 μg/mL and 80 μg/mL, respectively. And the LD₅₀ for Kunming mice of PEPB10 and PEPB25 were more than 5000 mg/kg, which mean that PEPB10 and PEPB25 with high hydrophilicity show low toxicity and better selectivity for R. solani. The cationic polymers can kill the R. solani by damaging their membranes and exchanging the Ca²⁺ or/and Mg²⁺ cations of their membranes or cell wall. These results help to understand the antifungal mechanism of low-toxic polymeric quaternary ammonium salts and highlight their potential application as highly selective fungicidal agents for controlling plant diseases.     

Millerenolides,sesquiterpene lactones from Milleria quinqueflora

The aerial parts of Milleria quinqueflora afforded 17 new germacranolides (five melampolides and 12 millerenolides), two ent-pimarene and two ent-kaurene derivatives as well as two alicyclic diterpenes and a galactoside. The structures were elucidated by NMR spectroscopy. The chemotaxonomic situation is discussed.     

A critical assessment of the use of lipophilic cations as membrane potential probes

A critical review has been made of the literature on the use of lipophilic cations, such as triphenylmethyl phosphonium (TPMP⁺) as membrane potential probes in prokaryotes, uekaryote organelles in vitro, and eukaryote cells. An ideal lipophilic cation should be capable of penetrating through a biological membrane and obey the Nernst equation between a membrane bound phase and its environment. Many different forms of the Nernst equation are presented, useful in the calculation equilibrium potentials of lipophilic cations across membranes. Lipophilic cations appear to behave as valid membrane potential probes in prokaryotes and eukaryote organelles in vitro and even in vivo although some technical difficulties may be involved. On the other hand in valid forms of the Nernst equation have often been used to calculate the equilibrium potential of lipophilic cations across the plasma membranes of eukaryotic cells. In particular, the problem of intracellular compartmentation of lipophilic cations has often not been appreciated. Lipophilic cations do not appear to behave as reliable plasma membrane potential probes in eukaryotic cells. Some other avenues are discussed which might be useful in the determination of the plasma membrane potentials of small eukaryotic cells, e.g. the use of lipophilic anions as membrane potential probes.     

More chemistry of the thaxtomin phytotoxins

Chemical and biochemical studies indicated the possible involvement of N-acetyltryptophan and 4-nitrotryptophan as intermediates in biosynthesis of the thaxtomin phytotoxins. A search for other potential pathways indirectly resulted in the identification of three unusual thaxtomin analogues derived from the o-thaxtomin A isomer. Investigations to resolve the identity of a previously described thaxtomin A di-glucoside were not supportive of the proposed structure.     

Muscarinic Cholinergic Excitation of Smooth Muscle: Signal Transduction and Single Cationic Channel Properties

Acetylcholine, the principal neurotransmitter of the parasympathetic nervous system, evokes smooth muscle excitation and contraction by acting at the muscarinic receptors which, in many tissues, including the gastrointestinal tract, are comprised of the M₂ and M₃ subtypes. The opening of ion channels selective for monovalent cations (e.g., Na⁺ and K⁺) is the major mechanism of cholinergic excitation. We have studied signal transduction pathways and single cationic channel properties using patch-clamp recording and Ca²⁺ imaging techniques in guinea-pig single ileal myocytes. Cationic channels were found to couple to both M₂ and M₃ receptors via the GTP-bound G_oα and phospholipase C activation, respectively. When these primarily signaling links are established, cationic channel opening can be further potentiated by membrane depolarization and an increase in the intracellular Ca²⁺ concentration. A strong synergism exists between the receptor occupancy by the agonist and intrinsic voltage dependence of the current as the former can effectively modulate the voltage range of cationic channel activation, while membrane depolarization produces a strong sensitizing effect. However, at potentials close to 0 mV ion flux is terminated by channel flickery block, while further depolarization induces long-lasting channel inactivation. Channel flicker is not caused by intracellular Mg²⁺, polyamines, or any other freely diffusible molecule and is confined to potentials around 0 mV irrespective of the driving force. Thus, it appears to be an intrinsic channel property of physiological importance as it improves conditions for the action potential discharge and propagation. Similarly, intracellular Ca²⁺-dependent facilitation of channel opening is counteracted by a slower desensitization. Further, the most intriguing negative control was discovered in the experiments whereby all cellular G proteins were non-selectively and persistently activated by GTPγS infusion, in which case, over time, carbachol instead of activation caused strong and almost irreversible inhibition of the cationic current. In cell-attached and outside-out membrane patches exposed to 50 μM carbachol or 200 μM internal GTPγS, the activity of three types of cationic channels was observed. They had dissimilar conductances (10, 50, and 130 pS), voltage dependence, and kinetics. The properties of the 50 pS channel are consistent with the whole-cell current behavior, at least when [Ca²⁺] _i is “clamped” at 100 nM. The voltage-independent component of the cationic conductance, which appears at higher levels of [Ca²⁺] _i , is likely mediated by the 130 pS channel, while the role of the 10 pS channel at present is unclear. Thus, smooth muscle cationic channels can uniquely detect and integrate many of the most important physiological signals such as the active conformation of two different muscarinic receptors, their associated G proteins and enzymes, as well as membrane potential and [Ca²⁺] _i levels. Moreover, some signals act in synergy, while most of them, depending on the intensity, can be either stimulatory or inhibitory.     

Profiling of Intracellular Metabolites: An Approach to Understanding the Characteristic Physiology of Mycobacterium leprae

Mycobacterium leprae is the causative agent of leprosy and also known to possess unique features such as inability to proliferate in vitro. Among the cellular components of M. leprae, various glycolipids present on the cell envelope are well characterized and some of them are identified to be pathogenic factors responsible for intracellular survival in host cells, while other intracellular metabolites, assumed to be associated with basic physiological feature, remain largely unknown. In the present study, to elucidate the comprehensive profile of intracellular metabolites, we performed the capillary electrophoresis-mass spectrometry (CE-MS) analysis on M. leprae and compared to that of M. bovis BCG. Interestingly, comparison of these two profiles showed that, in M. leprae, amino acids and their derivatives are significantly accumulated, but most of intermediates related to central carbon metabolism markedly decreased, implying that M. leprae possess unique metabolic features. The present study is the first report demonstrating the unique profiles of M. leprae metabolites and these insights might contribute to understanding undefined metabolism of M. leprae as well as pathogenic characteristics related to the manifestation of the disease.     

Geranyl and neryl triazole bisphosphonates as inhibitors of geranylgeranyl diphosphate synthase

When inhibitors of enzymes that utilize isoprenoid pyrophosphates are based on the natural substrates, a significant challenge can be to achieve selective inhibition of a specific enzyme. One element in the design process is the stereochemistry of the isoprenoid olefins. We recently reported preparation of a series of isoprenoid triazoles as potential inhibitors of geranylgeranyl transferase II but these compounds were obtained as a mixture of olefin isomers. We now have accomplished the stereoselective synthesis of these triazoles through the use of epoxy azides for the cycloaddition reaction followed by regeneration of the desired olefin. Both geranyl and neryl derivatives have been prepared as single olefin isomers through parallel reaction sequences. The products were assayed against multiple enzymes as well as in cell culture studies and surprisingly a Z-olefin isomer was found to be a potent and selective inhibitor of geranylgeranyl diphosphate synthase.     

Diffusion, Exclusion, and Specific Binding in a Large Channel: A Study of OmpF Selectivity Inversion

We find that moderate cationic selectivity of the general bacterial porin OmpF in sodium and potassium chloride solutions is inversed to anionic selectivity in concentrated solutions of barium, calcium, nickel, and magnesium chlorides. To understand the origin of this phenomenon, we consider several factors, which include the binding of divalent cations, electrostatic and steric exclusion of differently charged and differently sized ions, size-dependent hydrodynamic hindrance, electrokinetic effects, and significant “anionic” diffusion potential for bulk solutions of chlorides of divalent cations. Though all these factors contribute to the measured selectivity of this large channel, the observed selectivity inversion is mostly due to the following two. First, binding divalent cations compensates, or even slightly overcompensates, for the negative charge of the OmpF protein, which is known to be the main cause of cationic selectivity in sodium and potassium chloride solutions. Second, the higher anionic (versus cationic) transport rate expected for bulk solutions of chloride salts of divalent cations is the leading cause of the measured anionic selectivity of the channel. Interestingly, at high concentrations the binding of cations does not show any pronounced specificity within the divalent series because the reversal potentials measured in the series correlate well with the corresponding bulk diffusion potentials. Thus our study shows that, in contrast to the highly selective channels of neurophysiology that employ mostly the exclusion mechanism, quite different factors account for the selectivity of large channels. The elucidation of these factors is essential for understanding large channel selectivity and its regulation in vivo.     

Mechanism and Role of Divalent Cation Binding of Bacteriorhodopsin

Several observations have already suggested that the carboxyl groups are involved in the association of divalent cations with bacteriorhodopsin (Chang et al., 1985). Here we show that at least part of the protons released from deionized purple membrane (`blue membrane') samples when salt is added are from carboxyl groups. We find that the apparent pK of magnesium binding to purple membrane in the presence of 0.5 mM buffer is 5.85. We suggest this is the pK of the carboxyl groups shifted from their usual pK because of the proton concentrating effect of the large negative surface potential of the purple membrane. Divalent cations may interact with negatively charged sites on the surface of purple membrane through the surface potential and/or through binding either by individual ligands or by conformation-dependent chelation. We find that divalent cations can be released from purple membrane by raising the temperature. Moreover, purple membrane binds only about half as many divalent cations after bleaching. Neither of these operations is expected to decrease the surface potential and thus these experiments suggest that some specific conformation in purple membrane is essential for the binding of a substantial fraction of the divalent cations. Divalent cations in purple membrane can be replaced by monovalent, (Na⁺ and K⁺), or trivalent, (La⁺⁺⁺) cations. Flash photolysis measurements show that the amplitude of the photointermediate, O, is affected by the replacement of the divalent cations by other ions, especially by La⁺⁺⁺. The kinetics of the M photointermediate and light-induced H⁺ uptake are not affected by Na⁺ and K⁺, but they are drastically lengthened by La⁺⁺⁺ substitution, especially at alkaline pHs. We suggest that the surface charge density and thus the surface potential is controlled by divalent cation binding. Removal of the cations (to make deionized blue membrane) or replacement of them (e.g. La⁺⁺⁺-purple membrane) changes the surface potential and hence the proton concentration near the membrane surface. An increase in local proton concentration could cause the protonation of critical carboxyl groups, for example the counter-ion to the protonated Schiff's base, causing the red shift associated with the formation of both deionized and acid blue membrane. Similar explanations based on regulation of the surface proton concentration can explain many other effects associated with the association of different cations with bacteriorhodopsin.     

Widespread Head-to-Head Hydrocarbon Biosynthesis in Bacteria and Role of OleA

Previous studies identified the oleABCD genes involved in head-to-head olefinic hydrocarbon biosynthesis. The present study more fully defined the OleABCD protein families within the thiolase, α/β-hydrolase, AMP-dependent ligase/synthase, and short-chain dehydrogenase superfamilies, respectively. Only 0.1 to 1% of each superfamily represents likely Ole proteins. Sequence analysis based on structural alignments and gene context was used to identify highly likely ole genes. Selected microorganisms from the phyla Verucomicrobia, Planctomyces, Chloroflexi, Proteobacteria, and Actinobacteria were tested experimentally and shown to produce long-chain olefinic hydrocarbons. However, different species from the same genera sometimes lack the ole genes and fail to produce olefinic hydrocarbons. Overall, only 1.9% of 3,558 genomes analyzed showed clear evidence for containing ole genes. The type of olefins produced by different bacteria differed greatly with respect to the number of carbon-carbon double bonds. The greatest number of organisms surveyed biosynthesized a single long-chain olefin, 3,6,9,12,15,19,22,25,28-hentriacontanonaene, that contains nine double bonds. Xanthomonas campestris produced the greatest number of distinct olefin products, 15 compounds ranging in length from C₂₈ to C₃₁ and containing one to three double bonds. The type of long-chain product formed was shown to be dependent on the oleA gene in experiments with Shewanella oneidensis MR-1 ole gene deletion mutants containing native or heterologous oleA genes expressed in trans. A strain deleted in oleABCD and containing oleA in trans produced only ketones. Based on these observations, it was proposed that OleA catalyzes a nondecarboxylative thiolytic condensation of fatty acyl chains to generate a β-ketoacyl intermediate that can decarboxylate spontaneously to generate ketones.There is currently great interest in elucidating the means by which microbes produce nongaseous hydrocarbons for use as specialty chemicals and fuels (8, 40). While many details remain to be revealed, there appear to be several different pathways by which microbes biosynthesize long-chain hydrocarbons. The most studied of the pathways (16) involves the condensation of isoprene units to generate hydrocarbons with a multiple of five carbon atoms (C₁₀, C₁₅, C₂₀, etc.). A more obscure biosynthetic route is a reported decarbonylation of fatty aldehydes to generate a C_n−1 hydrocarbon chain (19). A third mechanism that has received some attention is what has been denoted head-to-head condensation of fatty acids (2, 3, 4, 8, 64). In this pathway, the hydrocarbons are described to arise from the formation of a carbon-to-carbon bond between the carboxyl carbon of one fatty acid and the α-carbon of another fatty acid (3). This condensation results in a particular type of hydrocarbon with chain lengths of C₂₃ to C₃₃ and containing one or more double bonds. One double bond involves the median carbon in the chain at the point of fatty acid condensation. An example of this overall biosynthetic pathway leading to the formation of specific C₂₉ olefinic hydrocarbon isomers from fatty acid precursors has been demonstrated in vivo (61, 63, 64) and in vitro (4, 8).The condensation, elimination of carbon dioxide, and loss of the other carboxyl group oxygen atoms likely require multiple enzyme-catalyzed reactions. Recent patent applications by L. Friedman et al. describe a role for three or four proteins in this biosynthetic pathway (18 September 2008, WO2008/113041; 4 December 2008, WO2008/147781). Most recently, Beller et al. demonstrated the requirement for three genes from Micrococcus luteus in the biosynthesis of long-chain olefins (8). That study also demonstrated in vitro production of olefins by recombinant proteins in the presence of crude cell extracts from Escherichia coli. In another study, Shewanella oneidensis strain MR-1 was shown to produce a head-to-head hydrocarbon (59). A cluster of four genes, oleABCD, was shown to be involved in olefin biosynthesis by that organism.While genetic and biochemical data have provided evidence for Ole proteins producing long-chain olefins in M. luteus and S. oneidensis, there are many outstanding details of the biosynthesis that remain to be elucidated. Moreover, the extent to which microbial and other species produce head-to-head olefins is unclear. A recent patent application by Friedman and Rude (WO2008/113041) presented tables listing genes homologous to the ole genes described by Beller et al. (8). However, the homologs identified included genes from mouse and tree frog, organisms not known to produce head-to-head hydrocarbons. Additionally, hydrocarbon biosynthetic genes from Arthrobacter sp. FB24 were claimed, and that strain was later shown not to produce hydrocarbons under identical conditions for which other Arthrobacter strains did (22). In that context, the present study closely examined the protein sequence families of Ole proteins and the configurations of putative ole genes within genomes to identify those most likely to be involved in head-to-head hydrocarbon biosynthesis. This was followed by experimental testing for the presence of long-chain head-to-head hydrocarbons in representative bacteria from diverse phyla. This study also found that, of closely related bacteria, some produce head-to-head hydrocarbons and others do not.A previous publication investigated in vitro olefin biosynthesis from myristyl-coenzyme A (CoA) (8). That study showed ketone and olefin biosynthesis in vitro and proposed a mechanism requiring the participation of ancillary proteins not encoded in the oleABCD gene cluster. The mechanism proposed fatty acyl oxidation to generate a β-keto acid that is the substrate for the OleA protein.In fact, different mechanisms have been suggested previously for the biosynthesis of head-to-head olefins (3; Friedman and Rude, WO2008/113041; Friedman and Da Costa, WO2008/147781), and different roles for the OleA protein have been proposed (8; Friedman and Rude, WO2008/113041; Friedman and Da Costa, WO2008/147781). It is not possible to deduce the olefinic biosynthetic pathway or individual reaction types based on protein sequence alignments alone, because this pathway is unique, differing markedly from isoprenoid or decarbonylation hydrocarbon biosynthesis pathways. Moreover, the individual Ole proteins are each homologous to proteins that collectively catalyze diverse reactions. In that context, we (i) initiated a more detailed study of the Ole protein superfamilies, (ii) identified likely olefin (ole) biosynthetic genes out of thousands of homologs, (iii) experimentally tested bacteria from different phyla for long-chain olefins, (iv) developed insights into the role of OleA in head-to-head olefin biosynthesis, and (v) propose an alternative mechanism for head-to-head condensation of fatty acyl groups.     

Interaction of Organic Cations with Organic Anion Transporters

Studies of the organic anion transporters (Oats) have focused mainly on their interactions with organic anionic substrates. However, as suggested when Oat1 was originally identified as NKT (Lopez-Nieto, C. E., You, G., Bush, K. T., Barros, E. J., Beier, D. R., and Nigam, S. K. (1997) J. Biol. Chem. 272, 6471–6478), since the Oats share close homology with organic cation transporters (Octs), it is possible that Oats interact with cations as well. We now show that mouse Oat1 (mOat1) and mOat3 and, to a lesser degree, mOat6 bind a number of “prototypical” Oct substrates, including 1-methyl-4-phenylpyridinium. In addition to oocyte expression assays, we have tested binding of organic cations to Oat1 and Oat3 in ex vivo assays by analyzing interactions in kidney organ cultures deficient in Oat1 and Oat3. We also demonstrate that mOat3 transports organic cations such as 1-methyl-4-phenylpyridinium and cimetidine. A pharmacophore based on the binding affinities of the tested organic cations for Oat3 was generated. Using this pharmacophore, we screened a chemical library and were able to identify novel cationic compounds that bound to Oat1 and Oat3. These compounds bound Oat3 with an affinity higher than the highest affinity compounds in the original set of prototypical Oct substrates. Thus, whereas Oat1, Oat3, and Oat6 appear to function largely in organic anion transport, they also bind and transport some organic cations. These findings could be of clinical significance, since drugs and metabolites that under normal physiological conditions do not bind to the Oats may undergo changes in charge and become Oat substrates during pathologic conditions wherein significant variations in body fluid pH occur.