Pyridine salvage and nicotinic acid conjugate synthesis in leaves of mangrove species

The metabolic fate of [carbonyl-¹⁴C]nicotinamide was surveyed in leaf disks of seven mangrove species, Bruguiera gymnorrhiza, Rhizophora stylosa, Kandeliaobovata, Sonneratia alba, Pemphis acidula, Lumnitzera racemosa and Avicennia marina, with and without 250 mM NaCl. Uptake of [¹⁴C]nicotinamide by leaf disks was stimulated by 250 mM NaCl in K. candel, R. stylosa, A. marina and L. racemosa. [Carbonyl-¹⁴C]nicotinamide was converted to nicotinic acid and was utilised for the synthesis of nucleotides and nicotinic acid conjugates. Formation of nicotinic acid by the deaminase reaction was rapid; there was little accumulation of nicotinamide in the disks 3 h after administration. Radioactivity from [carbonyl-¹⁴C]nicotinamide was incorporated into pyridine nucleotides (mainly NAD and NADP) in all mangrove leaves, and the rates varied from 2% (in L. racemosa) to 15% (S. alba) of the total radioactivity taken up. NaCl generally reduced nicotinic acid salvage for NAD and NADP. In all mangrove leaf disks, the most heavily labelled compounds (up to 70% of total radioactivity) were trigonelline (N-methylnicotinic acid) and/or nicotinic acid N-glucoside. Trigonelline was formed in all mangrove plants, but N-glucoside synthesis was found only in leaves of A. marina and K. obovata. In A. marina, incorporation of radioactivity into N-glucoside (51%) was much greater than incorporation into trigonelline (2%). In general, NaCl stimulates the synthesis of these pyridine conjugates. The rate of decarboxylation of nicotinic acid in roots of A. marina seedlings was much greater than for the corresponding reaction observed in leaves.     

Production of shikimic acid

Shikimic acid is a key intermediate for the synthesis of the antiviral drug oseltamivir (Tamiflu®). Shikimic acid can be produced via chemical synthesis, microbial fermentation and extraction from certain plants. An alternative production route is via biotransformation of the more readily available quinic acid. Much of the current supply of shikimic acid is sourced from the seeds of Chinese star anise (Illicium verum). Supply from star anise seeds has experienced difficulties and is susceptible to vagaries of weather. Star anise tree takes around six-years from planting to bear fruit, but remains productive for long. Extraction and purification from seeds are expensive. Production via fermentation is increasing. Other production methods are too expensive, or insufficiently developed. In the future, production in recombinant microorganisms via fermentation may become established as the preferred route. Methods for producing shikimic acid are reviewed.     

Crystal structure of grape dihydroflavonol 4-reductase, a key enzyme in flavonoid biosynthesis

The nicotinamide adenine dinucleotide phosphate (NADPH)-dependent enzyme dihydroflavonol 4-reductase (DFR) catalyzes a late step in the biosynthesis of anthocyanins and condensed tannins, two flavonoid classes of importance to plant survival and human nutrition. This enzyme has been widely investigated in many plant species, but little is known about its structural and biochemical properties. To provide a basis for detailed structure-function studies, the crystal structure of Vitis vinifera DFR, heterologously expressed in Escherichia coli, has been determined at 1.8 Å resolution. The 3D structure of the ternary complex obtained with the oxidized form of nicotinamide adenine dinucleotide phosphate and dihydroquercetin, one of the DFR substrates, presents common features with the short-chain dehydrogenase/reductase family, i.e., an N-terminal domain adopting a Rossmann fold and a variable C-terminal domain, which participates in substrate binding. The structure confirms the importance of the 131-156 region, which lines the substrate binding site and enlightens the role of a specific residue at position 133 (Asn or Asp), assumed to control substrate recognition. The activity of the wild-type enzyme and its variant N133D has been quantified in vitro, using dihydroquercetin or dihydrokaempferol. Our results demonstrate that position 133 cannot be solely responsible for the recognition of the B-ring hydroxylation pattern of dihydroflavonols.     

Mechanistic analysis of allosteric and non-allosteric effects arising from nanobody binding to two epitopes of the dihydrofolate reductase of Escherichia coli

Although allosteric effector antibodies are used widely as modulators of receptors and enzymes, experimental analysis of their mechanism remains highly challenging. Here, we investigate the molecular mechanisms of allosteric and non-allosteric effector antibodies in an experimentally tractable system, consisting of single-domain antibodies (nanobodies) that target the model enzyme dihydrofolate reductase (DHFR) from Escherichia coli. A panel of thirty-five nanobodies was isolated using several strategies to increase nanobody diversity. The nanobodies exhibit a variety of effector properties, including partial inhibition, strong inhibition and stimulation of DHFR activity. Despite these diverse effector properties, chemical shift perturbation NMR epitope mapping identified only two epitope regions: epitope α is a new allosteric site that is over 10 Å from the active site, while epitope β is located in the region of the Met20 loop. The structural basis for DHFR allosteric inhibition or activation upon nanobody binding to the α epitope was examined by solving the crystal structures of DHFR in complex with Nb113 (an allosteric inhibitor) and Nb179 (an allosteric activator). The structures suggest roles for conformational constraint and altered protein dynamics, but not epitope distortion, in the observed allosteric effects. The crystal structure of a β epitope region binder (ca1698) in complex with DHFR is also reported. Although CDR3 of ca1698 occupies the substrate binding site, ca1698 displays linear mixed inhibition kinetics instead of simple competitive inhibition kinetics. Two mechanisms are proposed to account for this apparent anomaly. Evidence for structural convergence of ca1698 and Nb216 during affinity maturation is also presented.     

Molecular cloning and catalytic characterization of a recombinant tropine biosynthetic tropinone reductase from Withania coagulans leaf

Tropinone reductases (TRs) are small proteins belonging to the SDR (short chain dehydrogenase/reductase) family of enzymes. TR-I and TR-II catalyze the conversion of tropinone into tropane alcohols (tropine and pseudotropine, respectively). The steps are intermediary enroute to biosynthesis of tropane esters of medicinal importance, hyoscyamine/scopolamine, and calystegins, respectively. Biosynthesis of tropane alkaloids has been proposed to occur in roots. However, in the present report, a tropine forming tropinone reductase (TR-I) cDNA was isolated from the aerial tissue (leaf) of a medicinal plant, Withania coagulans. The ORF was deduced to encode a polypeptide of 29.34 kDa. The complete cDNA (WcTRI) was expressed in E. coli and the recombinant His-tagged protein was purified for functional characterization. The enzyme had a narrow pH range of substantial activity with maxima at 6.6. Relatively superior thermostability of the enzyme (30% retention of activity at 60 °C) was catalytic novelty in consonance with the desert area restricted habitat of the plant. The in vitro reaction kinetics predominantly favoured the forward reaction. The enzyme had wide substrate specificity but did not cover the substrates of other well-known plant SDR related to menthol metabolism. To our knowledge, this pertains to be the first report on any gene and enzyme of secondary metabolism from the commercially and medicinally important vegetable rennet species.     

Analysis of the domain properties of the novel cytochrome P450 RhF

The properties of the heme, flavin mononucleotide (FMN) and FeS domains of P450 RhF, from Rhodococcus sp. NCIMB 9784, expressed separately and in combination are analysed. The nucleotide preference, imidazole binding and reduction potentials of the heme and FMN domains are unaltered by their separation. The intact enzyme is monomeric and the flavin is confirmed to be FMN. The two one-electron reduction potentials of the FMN are -240 and -270 mV. The spectroscopic and thermodynamic properties of the FeS domain, masked in the intact enzyme, are revealed for the first time, confirming it as a 2Fe-2S ferredoxin with a reduction potential of -214 mV.     

Protection against reactive oxygen species by selenoproteins

Reactive oxygen species (ROS) are derived from cellular oxygen metabolism and from exogenous sources. An excess of ROS results in oxidative stress and may eventually cause cell death. ROS levels within cells and in extracellular body fluids are controlled by concerted action of enzymatic and non-enzymatic antioxidants. The essential trace element selenium exerts its antioxidant function mainly in the form of selenocysteine residues as an integral constituent of ROS-detoxifying selenoenzymes such as glutathione peroxidases (GPx), thioredoxin reductases (TrxR) and possibly selenoprotein P (SeP). In particular, the dual role of selenoprotein P as selenium transporter and antioxidant enzyme is highlighted herein. A cytoprotective effect of selenium supplementation has been demonstrated for various cell types including neurons and astrocytes as well as endothelial cells. Maintenance of full GPx and TrxR activity by adequate dietary selenium supply has been proposed to be useful for the prevention of several cardiovascular and neurological disorders. On the other hand, selenium supplementation at supranutritional levels has been utilised for cancer prevention: antioxidant selenoenzymes as well as prooxidant effects of selenocompounds on tumor cells are thought to be involved in the anti-carcinogenic action of selenium.     

Farnesol attenuates 1,2-dimethylhydrazine induced oxidative stress, inflammation and apoptotic responses in the colon of Wistar rats

Colon cancer is the major health hazard related with high mortality and it is a pathological consequence of persistent oxidative stress and inflammation. Farnesol, an isoprenoid alcohol, has been shown to possess antioxidant, anti-inflammatory and chemopreventive properties. The present study was performed to evaluate the protective efficacy of farnesol against 1,2-dimethylhydrazine (DMH) induced oxidative stress, inflammatory response and apoptotic tissue damage. Farnesol was administered once daily for seven consecutive days at the doses of 50 and 100 mg/kg body weight in corn oil. On day 7, a single injection of DMH was given subcutaneously in the groin at the dose of 40 mg/kg body weight. Protective effects of farnesol were assessed by using caspase-3 activity, tissue lipid peroxidation (LPO) and antioxidant status as end point markers. Further strengthening was evident on histopathological observations used to assess the protective efficacy of farnesol. Prophylactic treatment with farnesol significantly ameliorates DMH induced oxidative damage by diminishing the tissue LPO accompanied by increase in enzymatic viz., superoxide dismutase (SOD), catalase, glutathione peroxidase (GPx), glutathione reductase (GR), glutathione-S-transferase (GST) and quinone reductase (QR) and non-enzymatic viz., reduced glutathione (GSH) antioxidant status. Farnesol supplementation significantly decreased caspase-3 activity in colonic tissue. Histological findings also revealed that pretreatment with farnesol significantly reduced the severity of submucosal edema, regional destruction of the mucosal layer and intense infiltration of the inflammatory cells in mucosal and submucosal layers of the colon. The data of the present study suggest that farnesol effectively suppress DMH induced colonic mucosal damage by ameliorating oxidative stress, inflammatory and apoptotic responses.     

In vitro effects of active constituents and extracts of Orthosiphon stamineus on the activities of three major human cDNA-expressed cytochrome P450 enzymes

Orthosiphon stamineus (OS) has been traditionally used to treat diabetes, kidney and urinary disorders, high blood pressure and bone or muscular pain. To assess the possibility of drug–herb interaction via interference of metabolism, effects of four OS extracts of different polarity and three active constituents (sinensetin, eupatorin and rosmarinic acid) on major human cDNA-expressed cytochrome P450 (CYP) enzymes were investigated. Three substrate-probe based high-performance liquid chromatography (HPLC) assays were established to serve as activity markers for CYP2C9, CYP2D6 and CYP3A4. Our results indicate that OS extracts and constituents exhibited differential modulatory effects on different CYPs. While none of the OS components showed significant inhibition on CYP2C9, eupatorin strongly and uncompetitively inhibited CYP2D6 activity with a K_i value of 10.2 μM. CYP3A4 appeared to be the most susceptible enzyme to OS inhibitory effects. It was moderately inhibited by OS dichloromethane and petroleum ether extract with mixed-type and noncompetitive inhibitions (K_i = 93.7 and 44.9 μg/mL), respectively. Correlation study indicated that the inhibition was accounted for by the presence of eupatorin in the extracts. When IC₅₀ values of these extracts were expressed in volume per dose unit to reflect inhibitory effect at recommended human doses from commercially available products, moderate inhibition was also observed. In addition, CYP3A4 was strongly and noncompetitively inhibited by eupatorin alone, with a K_i value of 9.3 μM. These findings suggest that co-administration of OS products, especially those with high eupatorin content, with conventional drugs may have the potential to cause drug–herb interactions involving inhibition of major CYP enzymes.     

Purification and characterization of trehalose phosphorylase from the commercial mushroom Agaricus bisporus

Trehalose phosphorylase (EC 2.4.1.64) from Agaricus bisporus was purified for the first time from a fungus. This enzyme appears to play a key role in trehalose metabolism in A. bisporus since no trehalase or trehalose synthase activities could be detected in this fungus. Trehalose phosphorylase catalyzes the reversible reaction of degradation (phosphorolysis) and synthesis of trehalose. The native enzyme has a molecular weight of 240 kDa and consists of four identical 61-kDa subunits. The isoelectric point of the enzyme was pH 4.8. The optimum temperature for both enzyme reactions was 30°C. The optimum pH ranges for trehalose degradation and synthesis were 6.0–7.5 and 6.0–7.0, respectively. Trehalose degradation was inhibited by ATP and trehalose analogs, whereas the synthetic activity was inhibited by P_i (K_i=2.0 mM). The enzyme was highly specific towards trehalose, P_i, glucose and α-glucose-1-phosphate. The stoichiometry of the reaction between trehalose, P_i, glucose and α-glucose-1-phosphate was 1:1:1:1 (molar ratio). The K_m values were 61, 4.7, 24 and 6.3 mM for trehalose, P_i, glucose and α-glucose-1-phosphate, respectively. Under physiological conditions, A. bisporus trehalose phosphorylase probably performs both synthesis and degradation of trehalose.     

Characterization of two different acyl carrier proteins in complex I from Yarrowia lipolytica

Acyl carrier proteins of mitochondria (ACPMs) are small (∼ 10 kDa) acidic proteins that are homologous to the corresponding central components of prokaryotic fatty acid synthase complexes. Genomic deletions of the two genes ACPM1 and ACPM2 in the strictly aerobic yeast Yarrowia lipolytica resulted in strains that were not viable or retained only trace amounts of assembled mitochondrial complex I, respectively. This suggested different functions for the two proteins that despite high similarity could not be complemented by the respective other homolog still expressed in the deletion strains. Remarkably, the same phenotypes were observed if just the conserved serine carrying the phosphopantethein moiety was exchanged with alanine. Although this suggested a functional link to the lipid metabolism of mitochondria, no changes in the lipid composition of the organelles were found. Proteomic analysis revealed that both ACPMs were tightly bound to purified mitochondrial complex I. Western blot analysis revealed that the affinity tagged ACPM1 and ACPM2 proteins were exclusively detectable in mitochondrial membranes but not in the mitochondrial matrix as reported for other organisms. Hence we conclude that the ACPMs can serve all their possible functions in mitochondrial lipid metabolism and complex I assembly and stabilization as subunits bound to complex I.     

Action of halothane upon mitochondrial respiration

The inhibitory action of halothane upon respiration was studied with rat liver mitochondria (RLM³), beef heart mitochondria (HBHM), and electron-transport particles (ETP). With intact mitochondrial preparations the oxidation of NADH-linked substrates but not of succinate was markedly suppressed by low concentrations of halothane (<2 mm as determined by gas-liquid chromatography). This inhibitory action of halothane was completely reversible. In contrast, a number of other mitochondrial processes were found to be sensitive in an irreversible manner at higher concentrations of the anesthetic. Likewise, the oxidation of added NADH by HBHM, ETP, and detergent-disrupted RLM was found to be sensitive in a reversible manner to low concentrations of halothane. The energy-dependent transfer of electrons from succinate to NAD by ETP_H was also sensitive to halothane. On the other hand, the NADH-ferricyanide reductase and the succinic oxidase activities of ETP and the NADH-cytochrome c reductase activity of microsomes were all insensitive to halothane. The site of inhibition by halothane appears to be in the vicinity of the rotenone-sensitive site of complex I (NADH-CoQ reductase). A number of other general anesthetics inhibited respiration at or near the same site as halothane.     

Structural and Functional Characterization of Plant Aminoaldehyde Dehydrogenase from Pisum sativum with a Broad Specificity for Natural and Synthetic Aminoaldehydes

Aminoaldehyde dehydrogenases (AMADHs, EC 1.2.1.19) belong to the large aldehyde dehydrogenase (ALDH) superfamily, namely, the ALDH9 family. They oxidize polyamine-derived ω-aminoaldehydes to the corresponding ω-amino acids. Here, we report the first X-ray structures of plant AMADHs: two isoenzymes, PsAMADH1 and PsAMADH2, from Pisum sativum in complex with β-nicotinamide adenine dinucleotide (NAD⁺) at 2.4 and 2.15 Å resolution, respectively. Both recombinant proteins are dimeric and, similarly to other ALDHs, each monomer is composed of an oligomerization domain, a coenzyme binding domain and a catalytic domain. Each subunit binds NAD⁺ as a coenzyme, contains a solvent-accessible C-terminal peroxisomal targeting signal (type 1) and a cation bound in the cavity close to the NAD⁺ binding site. While the NAD⁺ binding mode is classical for PsAMADH2, that for PsAMADH1 is unusual among ALDHs. A glycerol molecule occupies the substrate binding site and mimics a bound substrate. Structural analysis and substrate specificity study of both isoenzymes in combination with data published previously on other ALDH9 family members show that the established categorization of such enzymes into distinct groups based on substrate specificity is no more appropriate, because many of them seem capable of oxidizing a large spectrum of aminoaldehyde substrates. PsAMADH1 and PsAMADH2 can oxidize N,N,N-trimethyl-4-aminobutyraldehyde into γ-butyrobetaine, which is the carnitine precursor in animal cells. This activity highly suggests that in addition to their contribution to the formation of compatible osmolytes such as glycine betaine, β-alanine betaine and γ-aminobutyric acid, AMADHs might participate in carnitine biosynthesis in plants.     

Effects of osmotic pretreatments on oxidative stress, antioxidant profiles and cryopreservation of olive somatic embryos

A three-day pretreatment of olive somatic embryos (SE) with 0.75 M sucrose, combined with cryoprotection (0.5 M DMSO, 1 M sucrose, 0.5 M glycerol and 0.009 M proline) and controlled rate cooling, supported regrowth (as 34.6% fresh weight gain) and resumption of embryo development after cryopreservation. Pretreatment with mannitol or sorbitol did not support regrowth. Profiles of sugars, proline, antioxidant enzymes, Reactive oxygen species (ROS), secondary oxidation products and ethylene were constructed for the most successful (0.75 M) pretreatment series. Sucrose was the optimal pretreatment for supporting recovery, it also elevated glutathione reductase (GR) activity compared to controls, whereas superoxide dismutase (SOD), catalase and guaiacol peroxidase activities remained relatively unchanged. Superoxide dismutase activity was higher in SE pretreated with sucrose, compared with those pretreated with polyols; H₂O₂ was enhanced in SE pretreated with sorbitol and sucrose compared to mannitol. The overall trend for ethylene and OH production revealed their levels were highest in SE pretreated with polyols albeit, for individual treatments this was not always the case. Generally, pretreatments did not significantly change embryo secondary oxidation profiles of ThioBarbituric Acid Reactive Substances (TBARS) and Schiff's bases. In combination these studies suggest oxidative processes may influence regrowth of cryopreserved olive SE and that optimal pretreatments could, in part, increase tolerance by an overall enhancement of endogenous antioxidants (particularly GR), proline and sugars.     

The regulation of xanthophyll cycle activity and of non-photochemical fluorescence quenching by two alternative electron flows in the diatoms Phaeodactylum tricornutum and Cyclotella meneghiniana

Intact cells of diatoms are characterized by a rapid diatoxanthin epoxidation during low light periods following high light illumination while epoxidation is severely restricted in phases of complete darkness. The present study shows that rapid diatoxanthin epoxidation is dependent on the availability of the cofactor of diatoxanthin epoxidase, NADPH, which cannot be generated in darkness due to the inactivity of PSI. In the diatom Phaeodactylum tricornutum, NADPH production during low light is dependent on PSII activity, and addition of DCMU consequently abolishes diatoxanthin epoxidation. In contrast to P. tricornutum, DCMU does not affect diatoxanthin epoxidation in Cyclotella meneghiniana, which shows the same rapid epoxidation in low light both in the absence or presence of DCMU. Measurements of the reduction state of the PQ pool and PSI activity indicate that, in the presence of DCMU, NADPH production in C. meneghiniana occurs via alternative electron transport, which includes electron donation from the chloroplast stroma to the PQ pool and, in a second step, from PQ to PSI. Similar electron flow to PQ is also observed during high light illumination of DCMU-treated P. tricornutum cells. In contrast to C. meneghiniana, the electrons are not directed to PSI, but most likely to a plastoquinone oxidase. This chlororespiratory electron transport leads to the establishment of an uncoupler-sensitive proton gradient in the presence of DCMU, which induces diadinoxanthin de-epoxidation and NPQ. In C. meneghiniana, electron flow to the plastoquinone oxidase is restricted, and consequently, diadinoxanthin de-epoxidation and NPQ is not observed after addition of DCMU.     

Purification, characterization, and directed evolution study of a vitamin D3 hydroxylase from Pseudonocardia autotrophica

Vitamin D₃ (VD₃) is a fat-soluble prohormone that plays a crucial role in bone metabolism, immunity, and control of cell proliferation and cell differentiation in mammals. The actinomycete Pseudonocardia autotrophica is capable of bioconversion of VD₃ into its physiologically active forms, namely, 25(OH)VD₃ or 1α,25(OH)₂VD₃. In this study, we isolated and characterized Vdh (vitamin D₃ hydroxylase), which hydroxylates VD₃ from P. autotrophica NBRC 12743. The vdh gene encodes a protein containing 403 amino acids with a molecular weight of 44,368 Da. This hydroxylase was found to be homologous with the P450 belonging to CYP107 family. Vdh had the same ratio of the V_max values for VD₃ 25-hydroxylation and 25(OH)VD₃ 1α-hydroxylation, while other enzymes showed preferential regio-specific hydroxylation on VD₃. We characterized a collection of Vdh mutants obtained by random mutagenesis and obtained a Vdh-K1 mutant by the combination of four amino acid substitutions. Vdh-K1 showed one-order higher VD₃ 25-hydroxylase activity than the wild-type enzyme. Biotransformation of VD₃ into 25(OH)VD₃ was successfully accomplished with a Vdh-expressed recombinant strain of actinobacterium Rhodococcus erythropolis. Vdh may be a useful enzyme for the production of physiologically active forms of VD₃ by a single cytochrome P450.     

A Tris-induced change in the midpoint potential of Z,the donor to photosystem II,as determined by the kinetics of the back reaction

A new method of measuring the rate of the back reaction from the state Z⁺ P680 Q_A^? in Tris-washed chloroplasts is described. By using ratios of back reaction rates we demonstrate a Tris-induced change in the equilibrium between Z and P680 and attribute this change to an alteration of the midpoint potential of Z by Tris treatment. We also demonstrate that the previously observed inhibition of the back reaction by ADRY reagents can be localized at Z and understood in terms of electron donation to Z⁺ by ADRY reagents.