Final Reduction Step Research Articles

The phenylpropene synthase pathway and its applications in the engineering of volatile phenylpropanoids in plants

Phenylpropenes such as eugenol, isoeugenol, chavicol, and anethole are C6–C3 volatile compounds derived from phenylalanine by modification of its benzene ring and reduction of its propyl side chain, with the final reduction step catalyzed by a product-specific phenylpropene synthase. Recent advances in the biochemical and molecular analysis of phenylpropene synthases have improved our understanding of their evolution, structural properties, and reaction mechanism, providing insights into how plants produce and regulate the many types of phenylpropene volatiles. Since phenylpropenes are important in determining the flavor of foods and the quality of essential oils in cosmetics, the identification of the genes and enzymes responsible for the biosynthesis of phenylpropene volatiles has also provided us with tools to meet the challenge of improving plant aromas through genetic engineering.

Completing our understanding of tetracycline biosynthesis: the enzymatic basis of the F420‐dependent final reduction step.

Tetracyclines are important broad‐spectrum antibiotics. While the discovery of tetracyclines date back to the 1940s, our understanding of the biosynthesis of tetracyclines has only been investigated in recent years. One unknown step in the pathway is the conversion of 5a, 11a‐dehydrotetracycline to either oxytetracycline in Streptomyces rimosus or tetracycline in Streptomyces aureofaciens. The reduction of the 5a–11a double bond is likely catalyzed by a F420‐dependent enzymes, however, the enzymatic basis for this reaction remains unresolved. Using a heterologous expression platform as a starting point, we discovered a small, previously unknown enzyme in both the oxy and ctc pathways that is responsible for this reduction. In vitro reconstitution using this small enzymes, in conjunction with the anhydrotetracycline oxygenase, showed that anhydrotetracycline can be converted to the final products. We also provide biochemical and structural explanation for the differential formation of oxytetracycline and tetracycline from the respective pathways. The result of this work therefore completes our understanding of the tetracycline pathways and pave the way for engineered biosynthesis of analogs.

Synthesis of an <em>In vivo</em> MRI-detectable Apoptosis Probe

Cellular apoptosis is a prominent feature of many diseases, and this programmed cell death typically occurs before clinical manifestations of disease are evident. A means to detect apoptosis in its earliest, reversible stages would afford a pre-clinical 'window' during which preventive or therapeutic measures could be taken to protect the heart from permanent damage. We present herein a simple and robust method to conjugate human Annexin V (ANX), which avidly binds to cells in the earliest, reversible stages of apoptosis, to superparamagnetic iron oxide (SPIO) nanoparticles, which serve as an MRI-detectable contrast agent. The conjugation method begins with an oxidation of the SPIO nanoparticles, which oxidizes carboxyl groups on the polysaccharide shell of SPIO. Purified ANX protein is then added in the setting of a sodium borate solution to facilitate covalent interaction of ANX with SPIO in a reducing buffer. A final reduction step with sodium borohydride is performed to complete the reduction, and then the reaction is quenched. Unconjugated ANX is removed from the mix by microcentrifuge filtration. The size and purity of the ANX-SPIO product is verified by dynamic light scattering (DLS). This method does not require addition to, or modification of, the polysaccharide SPIO shell, as opposed to cross-linked iron oxide particle conjugation methods or biotin-labeled nanoparticles. As a result, this method represents a simple, robust approach that may be extended to conjugation of other proteins of interest.

On the rate-determining step and the ligand electronic effects in rhodium catalysed hydrogenation of enamines and the hydroaminomethylation of alkenes

In the course of studies on the tandem hydroformylation–reductive amination (hydroaminomethylation), fluorinated mono-phosphines were found to be more active than their more electron-donating counterparts in the enamine hydrogenation step of the reaction; this is in contrast to the widely held view that alkene hydrogenation activity increases with ligand donor strength. DFT calculations comparing the reaction pathways for a simple alkene and a representative enamine show that the rate-determining step changes from the first insertion into a Rh–H bond for but-2-ene to the final reductive elimination step from the Rh–hydride–alkyl species in the enamine hydrogenations.

Coxiella burnetii Expresses a Functional Δ24 Sterol Reductase

Coxiella burnetii, the etiological agent of human Q fever, occupies a unique niche inside the host cell, where it replicates in a modified acidic phagolysosome or parasitophorous vacuole (PV). The PV membrane is cholesterol-rich, and inhibition of host cholesterol metabolism negatively impacts PV biogenesis and pathogen replication. The precise source(s) of PV membrane cholesterol is unknown, as is whether the bacterium actively diverts and/or modifies host cell cholesterol or sterol precursors. C. burnetii lacks enzymes for de novo cholesterol biosynthesis; however, the organism encodes a eukaryote-like Δ24 sterol reductase homolog, CBU1206. Absent in other prokaryotes, this enzyme is predicted to reduce sterol double bonds at carbon 24 in the final step of cholesterol or ergosterol biosynthesis. In the present study, we examined the functional activity of CBU1206. Amino acid alignments revealed the greatest sequence identity (51.7%) with a Δ24 sterol reductase from the soil amoeba Naegleria gruberi. CBU1206 activity was examined by expressing the protein in a Saccharomyces cerevisiae erg4 mutant under the control of a galactose-inducible promoter. Erg4 is a yeast Δ24 sterol reductase responsible for the final reduction step in ergosterol synthesis. Like Erg4-green fluorescent protein (GFP), a CBU1206-GFP fusion protein localized to the yeast endoplasmic reticulum. Heterologous expression of CBU1206 rescued S. cerevisiae erg4 sensitivity to growth in the presence of brefeldin A and cycloheximide and resulted in new synthesis of ergosterol. These data indicate CBU1206 is an active sterol reductase and suggest the enzyme may act on host sterols during C. burnetii intracellular growth.

Tetrahydrolipstatin Inhibition, Functional Analyses, and Three-dimensional Structure of a Lipase Essential for Mycobacterial Viability

The highly complex and unique mycobacterial cell wall is critical to the survival of Mycobacteria in host cells. However, the biosynthetic pathways responsible for its synthesis are, in general, incompletely characterized. Rv3802c from Mycobacterium tuberculosis is a partially characterized phospholipase/thioesterase encoded within a genetic cluster dedicated to the synthesis of core structures of the mycobacterial cell wall, including mycolic acids and arabinogalactan. Enzymatic assays performed with purified recombinant proteins Rv3802c and its close homologs from Mycobacterium smegmatis (MSMEG_6394) and Corynebacterium glutamicum (NCgl2775) show that they all have significant lipase activities that are inhibited by tetrahydrolipstatin, an anti-obesity drug that coincidently inhibits mycobacterial cell wall biosynthesis. The crystal structure of MSMEG_6394, solved to 2.9 Å resolution, revealed an α/β hydrolase fold and a catalytic triad typically present in esterases and lipases. Furthermore, we demonstrate direct evidence of gene essentiality in M. smegmatis and show the structural consequences of loss of MSMEG_6394 function on the cellular integrity of the organism. These findings, combined with the predicted essentiality of Rv3802c in M. tuberculosis, indicate that the Rv3802c family performs a fundamental and indispensable lipase-associated function in mycobacteria.

ChemInform Abstract: Biosynthesis of Ansatrienin: Stereochemical Course of the Final Reduction Step Leading to the Cyclohexanecarboxylic Acid Moiety.

ChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 100 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a “Full Text” option. The original article is trackable via the “References” option.

A simple and fast method to analyze the orientation of c-type cytochromes in the outer membrane of Gram-negative bacteria

Outer membrane cytochromes catalyze the final reduction step of respiratory chains to electron acceptors that cannot diffuse through the outer membrane of Gram-negative bacteria. We developed an in vivo method to detect the orientation of outer membrane cytochromes via analysis of electron transfer reactions between these enzymes and riboflavin.

The presence of thiolated compounds allows the immobilization of enzymes on glyoxyl agarose at mild pH values: New strategies of stabilization by multipoint covalent attachment

Abstract Highly activated glyoxyl-supports rapidly immobilize proteins at pH 10 (where the ɛ-amino groups of the Lys groups of the protein surface are very reactive), and stabilize them by multipoint covalent attachment. However, they do not immobilize proteins at pH 8. This paper shows that the enzyme immobilization at this mild pH value is possible by incubation of the enzymes in the presence of different thiolated compounds (dithiothreitol, DTT; was selected as optimal reagent). The thiolated compounds (even the not reducing ones) stabilized the imino bonds formed at pH 8 between the aldehydes in the support and the amino groups of the protein. However, thiolated compounds are unable to reduce the imino bonds or the aldehyde groups and a final reduction step (e.g., using sodium borohydride) was always necessary. After enzyme immobilization through the most reactive amino group of the protein, the further incubation of this immobilized enzyme at pH 10 would improve the reactivity of the ɛ-amino groups of the Lys residues of the protein surface. Then, an intense multipoint covalent reaction of the enzyme with the dense layer of glyoxyl groups in the support could be obtained, increasing the stability of the immobilized enzyme. Using three different industrially relevant enzymes (penicillin G acylase from Escherichia coli (PGA), lipase from Bacillus thermocatenulatus (BTL2) and glutaryl acylase from Pseudomonas sp. (GA)), new immobilized-stabilized biocatalysts of the enzymes were produced. After reduction, the preparations incubated at pH 10 were more stable than those that were only immobilized and reduced at pH 8. In the case of the PGA, this preparation was even 4–5-fold more stable than those obtained by direct immobilization at pH 10 (around 40,000–50,000-fold more stable than the soluble enzyme).

Photoreduction of 2-methyl-1-nitro-9,10-anthraquinone in the presence of 1-phenylethanol

The photoreactions of 1-nitro-9,10-anthraquinone (N1) and 2-methyl-1-nitro-9,10-anthraquinone (N2) were studied in benzene and acetonitrile in the presence of 1-phenylethanol. For N2, a short-lived 10 ns transient observed upon flash photolysis is attributed to a triplet state, which can be intercepted by 1-phenylethanol to form a monohydro radical of N2 and a spectroscopically not detectable donor-derived radical. The decay of radicals yields the corresponding nitroso compound and eventually 1-amino-2-methyl-9,10-anthraquinone (A2) as photoproduct. The final reduction step requires participation of the anthraquinone carbonyl groups. The yield of radicals and the quantum yield (Phi(NH(2))) of conversion to A2 are small in inert solvents and increase with the donor concentration, approaching Phi(NH(2)) = 0.2. No triplet state was observed in the flash photolysis of N1, but a N1-derived radical and 1-amino-9,10-anthraquinone (A1) as final products were found. Various mechanistic aspects of complete photoreduction of nitroarenes to aminoarenes are discussed.

Selective hydrogenation of 4-(6-methoxy-2-naphtyl)-3-buten-2-one to Nabumetone ®

Low loading copper catalysts allows hydrogenation of α,β-unsaturated ketones to the corresponding saturated ones with selectivity up to 99%. Moreover, the selectivity can be tuned toward different products only by playing with the support acidity or the experimental conditions. By using a 8% Cu/SiO 2 catalyst the final reduction step in the synthesis of the anti-inflammatory drug Nabumetone ® can be carried out with selectivity >98% under mild conditions with good productivity and without any additives. No leaching of the metallic phase was observed, the catalyst is not pyrophoric even in the reduced state and it can be reused.

Type II Fatty Acid Biosynthesis, a New Approach in Antimalarial Natural Product Discovery

Malaria, one of the most problematic infectious diseases worldwide, is on the rise. The absence of an effective vaccine and the spread of drug-resistant strains of Plasmodium clearly indicate the necessity for the development of new chemotherapeutic agents and the identification of novel targets. The recent discovery of a relict, non-photosynthetic plastid-like organelle, the so-called apicoplast, in Plasmodium has opened up new avenues in malaria research. It also initiated the Plasmodium falciparum genome sequencing project, which revealed a number of biochemical pathways previously unknown to Plasmodium, i.e. cytosolic shikimate pathway, apicoplastic type II fatty acid, non-mevalonate isoprene and haem biosyntheses. Since these vital biosynthetic processes are absent in humans or fundamentally different from those found in humans, they represent excellent targets for pharmaceutical interventions. We are interested in the type II fatty acid synthase (FAS II) system of malaria parasite and focus on the FabI enzyme, the only known enoyl-ACP reductase in Plasmodium involved in the final reduction step of the fatty acid chain elongation cycle. Here we describe the general aspects of fatty acid biosynthesis, its essentiality to the malaria parasite and our continuing efforts to discover in Turkish medicinal plants natural antimalarial agents, which specifically target the plasmodial FabI enzyme.

Identification and cloning of the gene involved in the final step of chlortetracycline biosynthesis in Streptomyces aureofaciens.

For chlortetracycline biosynthesis in Streptomyces aureofaciens, the final reduction step is essential to give an antibiotic activity to its intermediate, which is catalyzed by tetracycline dehydrogenase with 7,8-dedimethyl-8-hydroxy-5-deazariboflavin (FO) as a cofactor. We identified and cloned the gene, which is essential for the biosynthesis of 6-demethyltetracycline and participates in the final step of its biosynthesis, from the genomic DNA of the 6-demethyltetracycline producer S. aureofaciens HP77. DNA sequence analysis revealed that the gene (tchA) had an open reading frame of 455 amino acids with an estimated molecular mass of 48.1 kDa. Southern hybridization analysis revealed that the tchA gene was located external to the chlortetracycline biosynthetic gene cluster in the genome. A conserved domain search of protein sequence databases indicated that TchA showed a similarity to FbiB, which is involved in the modification of FO in Mycobacterium bovis.

Diastereoselective Synthesis of Linear‐Fused Tricyclic Nitrogen Heterocycles by a Tandem Reduction‐Reductive Amination Reaction.

AbstractFor Abstract see ChemInform Abstract in Full Text.

Diastereoselective synthesis of linear‐fused tricyclic nitrogen heterocycles by a tandem reduction‐reductive amination reaction

AbstractA two‐step diastereoselective synthesis of linear‐fused tricyclic nitrogen heterocycles has been developed from cyclic β‐ketoesters. The cyclization substrates are readily prepared by alkylation of the methyl 2‐oxo‐cycloalkanecarboxylates with 2‐nitrobenzyl bromide. Hydrogenation of these substrates initiates a reaction sequence involving (1) reduction of the aromatic nitro group, (2) condensation of the resulting hydroxyl‐amine or aniline nitrogen with the cycloalkanone and (3) reduction of the imine. The products are isolated in high yield as single diastereomers having the trans‐fused ring junction. The observed selectivity is rationalized in terms of a steric effect imposed by the ester group in the final reductive amination step which directs the incoming hydrogen to the opposite face of the molecule. By comparison, reductive cyclizations of substrates lacking the stereodirecting ester group give mixtures of cis and trans products with a preference for the cis‐fused heterocycle.

Mechanism of the six-electron reduction of nitrite to ammonia by cytochrome c nitrite reductase.

Cytochrome c nitrite reductase catalyzes the six-electron reduction of nitrite to ammonia without the release of potential reaction intermediates, such as NO or hydroxylamine. On the basis of the crystallographic observation of reaction intermediates and of density functional calculations, we present a working hypothesis for the reaction mechanism of this multiheme enzyme which carries a novel lysine-coordinated heme group (Fe-Lys). It is proposed that nitrite reduction starts with a heterolytic cleavage of the N-O bond which is facilitated by a pronounced back-bonding interaction of nitrite coordinated through nitrogen to the reduced (Fe(II)) but not the oxidized (Fe(III)) active site iron. This step leads to the formation of an [FeNO](6) species and a water molecule and is further facilitated by a hydrogen bonding network that induces an electronic asymmetry in the nitrite molecule that weakens one N-O bond and strengthens the other. Subsequently, two rapid one-electron reductions lead to an [FeNO](8) form and, by protonation, to an Fe(II)-HNO adduct. Hereafter, hydroxylamine will be formed by a consecutive two-electron two-proton step which is dehydrated in the final two-electron reduction step to give ammonia and an additional water molecule. A single electron reduction of the active site closes the catalytic cycle.

Identification and characterization of 6-dehydroVB-A reductase from Streptomyces antibioticus

Streptomyces antibioticus NF-18 is a hyperproducing strain of a Streptomyces hormone, virginiae butanolide A (VB-A), that induces virginiamycin production of S. virginiae at nanomolar concentrations. To characterize the biosynthetic pathway of VB-A, we identified and characterized for the first time the 6-dehydroVB-A reductase that is responsible for the final reduction step in the biosynthesis. Assay protocols and stabilization conditions were established. The 6-dehydroVB-A reductase was found to require NADPH, not NADH, as a coenzyme. The K m values of the enzyme for NADPH and (±)-6-dehydroVB-A were determined to be 50±2 μM and 100±5 μM, respectively. Ultracentrifugation experiments revealed that 6-dehydroVB-A reductase was present almost exclusively in the 100 000× g supernatant fraction, indicating that the enzyme is a cytoplasmic-soluble protein. The M r of the native 6-dehydroVB-A reductase was estimated to be 82 000±3000 by molecular sieve HPLC. The optimal pH was found to be 6.7±0.2.

Identification and characterization of 6-dehydroVB-A reductase from Streptomyces antibioticus.

Streptomyces antibioticus NF-18 is a hyperproducing strain of a Streptomyces hormone, virginiae butanolide A (VB-A), that induces virginiamycin production of S. virginiae at nanomolar concentrations. To characterize the biosynthetic pathway of VB-A, we identified and characterized for the first time the 6-dehydro VB-A reductase that is responsible for the final reduction step in the biosynthesis. Assay protocols and stabilization conditions were established. The 6-dehydro VB-A reductase was found to require NADPH, not NADH, as a coenzyme. The K(m) values of the enzyme for NADPH and (+/-)-6-dehydro VB-A were determined to be 50 +/- 2 microM and 100 +/- 5 microM, respectively. Ultracentrifugation experiments revealed that 6-dehydro VB-A reductase was present almost exclusively in the 100,000 x g supernatant fraction, indicating that the enzyme is a cytoplasmic-soluble protein. The M(r) of the native 6-dehydro VB-A reductase was estimated to be 82,000 +/- 3000 by molecular sieve HPLC. The optimal pH was found to be 6.7 +/- 0.2.

Cloning and characterization of the gene encoding 1-cyclohexenylcarbonyl coenzyme A reductase from Streptomyces collinus.

We report the cloning of the gene encoding the 1-cyclohexenylcarbonyl coenzyme A reductase (ChcA) of Streptomyces collinus, an enzyme putatively involved in the final reduction step in the formation of the cyclohexyl moiety of ansatrienin from shikimic acid. The cloned gene, with a proposed designation of chcA, encodes an 843-bp open reading frame which predicts a primary translation product of 280 amino acids and a calculated molecular mass of 29.7 kDa. Highly significant sequence similiarity extending along almost the entire length of the protein was observed with members of the short-chain alcohol dehydrogenase superfamily. The S. collinus chcA gene was overexpressed in Escherichia coli by using a bacteriophage T7 transient expression system, and a protein with a specific ChcA activity was detected. The E. coli-produced ChcA protein was purified and shown to have similar steady-state kinetics and electrophoretic mobility on sodium dodecyl sulfate-polyacrylamide gels as the enoyl-coenzyme A reductase protein prepared from S. collinus. The enzyme demonstrated the ability to catalyze, in vitro, three of the reductive steps involved in the formation of cyclohexanecarboxylic acid. An S. collinus chcA mutant, constructed by deletion of a genomic region comprising the 5' end of chcA, lost the ChcA activity and the ability to synthesize either cyclohexanecarboxylic acid or ansatrienin. These results suggest that chcA encodes the ChcA that is involved in catalyzing multiple reductive steps in the pathway that provides the cyclohexanecarboxylic acid from shikimic acid.

Denitrification potential of soils from constructed and natural wetlands

Laboratory experiments were conducted to determine the NO 3 − removal (reduction) potential of wetland soils currently used for the disposal of reclaimed wastewater. Surface soil samples were collected, incubated under anoxic conditions with two levels of NO 3 −, and the evolution of N 2O was measured with time. Denitrification rates ranged from 0.06 to 0.92 g N m −2 day −1, when 10 mg NO 3-N per kg oil was added. Complete reduction of NO 3 − to N 2 occurred in the soils collected from two constructed wetlands (mineral soils) and one natural wetland (organic soil). However, the final reduction step was inhibited in the soils collected from two wetlands with organic soils. The inhibition was most likely the result of the lower pH of these soils. The results suggest that the addition of wastewater enhances the potential for denitrification in these soils.