Anthelmintic activity and non-cytotoxicity of phaeophorbide-a isolated from the leaf of Spondias mombin L.

ETHNOPHARMACOLOGICAL RELEVANCE: Helminthosis (worm infection) is a disease of grazing livestock, with significant economic implications. Increasing resistance to existing synthetic anthelmintics used to control helminthosis and the unwanted presence of residues of the anthelmintics reported in meat and dairy products present a serious global health challenge. These challenges have necessitated the development of novel anthelmintics that could combat drug resistance and exhibit better safety profiles. Spondias mombin L. (Anacardiaceae) is a plant that has been used traditionally as a worm expeller. AIM OF STUDY: The aim of the work reported herein was to isolate and characterise anthelmintic compound(s) from S. mombin leaf, establishing their bioactivity and safety profile. MATERIALS AND METHODS: Adult Haemonchus placei motility assay was used to assess anthelmintic bioactivity. Bioassay-guided chromatographic fractionation of acetone extract of S. mombin leaf was carried out on a silica gel stationary phase. The structure of the compound was elucidated using spectroscopy (1H and 13C NMR) and Liquid Chromatography-Mass Spectrometry (LC-ESI-MS). Screening to exclude potential cytotoxicity against mammalian cells (H460, Caco-2, MC3T3-E1) was done using alamar blue (AB) and CellTitreGlo (CTG) viability reagents. RESULTS: The acetone extract yielded an active fraction 8 (Ethyl acetate: methanol 90:10; anthelmintic LC50: 3.97 mg/mL), which yielded an active sub-fraction (Ethyl acetate: Methanol 95:5; anthelmintic LC50: 53.8 µg/mL), from which active compound 1 was isolated and identified as phaeophorbide-a (LC50: 23.0 µg/mL or 38.8 µM). The compound was not toxic below 200 µM but weakly cytotoxic at 200 µM. CONCLUSIONS: Phaeophorbide-a (1) isolated from S. mombin leaf extract and reported in the plant for the first time in this species demonstrated anthelmintic activity. No significant toxicity to mammalian cells was observed. It therefore represents a novel anthelmintic pharmacophore as a potential lead for the development of novel anthelmintics.

Re-opening the stage for Echinacea research - Characterization of phylloxanthobilins as a novel anti-oxidative compound class in Echinacea purpurea.

BACKGROUND: Phylloxanthobilins are tetrapyrrolic natural products that arise from the degradation of chlorophyll. Phylloxanthobilins have been discovered roughly 10 years ago in the leaves of deciduous trees, and are now considered a compound class with high and still unexplored potential of bioactivities. To date, however, there are no reports on the occurrence of phylloxanthobilins in parts of a medicinal plant used for pharmaceutical preparations. PURPOSE: The relevance of Echinacea purpurea as medicinal plant is undoubtedly high, and a large variety of pharmaceutical preparations is available on the market, mostly for the treatment of the common cold. Nevertheless, its phytochemical profiling has been limited to analysis for previously characterized substances, and this has not explained all its pharmacological efficacies. We therefore set out to investigate the occurrence of phylloxanthobilins in Echinacea purpurea. METHODS: Phylloxanthobilins in leaf extracts of Echinacea purpurea were detected using analytical HPLC. Identified phyllobilins were purified from plant material and characterized by UV/Vis, mass spectrometry, MS/MS, and confirmed by co-injections with previously published phyllobilins from different sources. The anti-oxidant activity of selected isolated phylloxanthobilins was assessed by an in vitro ferric reducing antioxidant power (FRAP) assay; in addition, the ability to scavenge ROS in cells caused by hydrogen peroxide stimulation was determined by measuring H2DCF-DA fluorescence and by assessing cellular GSH levels. RESULTS: In extracts of Echinacea purpurea leaves, an unprecedented diversity of phylloxanthobilins was detected; surprisingly, not only in senescent yellow leaves, but also in green leaves with no visible chlorophyll degradation. Six phylloxanthobilins were identified and structurally characterized. The uptake of phylloxanthobilins by human endothelial kidney cells was demonstrated. When investigating the anti-oxidative activity of these natural products, a potent in vitro activity was demonstrated; in addition, phylloxanthobilins possess intracellular ROS scavenging ability and can prevent oxidative stress as assessed by total cellular GSH levels. CONCLUSION: Phylloxanthobilins are important constituents of Echinacea purpurea extracts, and our first exploratory studies hint towards promising bioactivities of these natural products, which may be relevant for understanding Echinacea efficacies.

Yellow Dioxobilin-Type Tetrapyrroles from Chlorophyll Breakdown in Higher Plants-A New Class of Colored Phyllobilins.

In senescent leaves chlorophyll (Chl) catabolites typically accumulate as colorless tetrapyrroles, classified as formyloxobilin-type (or type-I) or dioxobilin-type (type-II) phyllobilins (PBs). Yellow type-I Chl catabolites (YCCs) also occur in some senescent leaves, in which they are generated by oxidation of colorless type-I PBs. A yellow type-II PB was recently proposed to occur in extracts of fall leaves of grapevine (Vitis vinifera), tentatively identified by its mass and UV/Vis absorption characteristics. Here, the first synthesis of a yellow type-II Chl catabolite (DYCC) from its presumed natural colorless type-II precursor is reported. A homogenate of a Spatiphyllum wallisii leaf was used as "green" means of effective and selective oxidation. The synthetic DYCC was fully characterized and identified with the yellow grapevine leaf pigment. As related yellow type-I PBs do, the DYCC functions as a reversible photoswitch by undergoing selective photo-induced Z/E isomerization of its C15=C16 bond.

Stereo- and regioselective phyllobilane oxidation in leaf homogenates of the peace lily (Spathiphyllum wallisii): hypothetical endogenous path to yellow chlorophyll catabolites.

In senescent leaves, chlorophyll typically is broken down to colorless and essentially photo-inactive phyllobilanes, which are linear tetrapyrroles classified as "nonfluorescent" chlorophyll catabolites (NCCs) and dioxobilane-type NCCs (DNCCs). In homogenates of senescent leaves of the tropical evergreen Spathiphyllum wallisii, when left at room temperature and extracted with methanol, the major endogenous, naturally formed NCC was regio- and stereoselectively oxidized (in part) to a mixture of its 15-hydroxy and 15-methoxy derivative. In the absence of methanol in the extract, only the 15-OH-NCC was observed. The endogenous oxidation process depended upon molecular oxygen. It was inhibited by carbon monoxide, as well as by keeping the leaf homogenate and extract at low temperatures. The remarkable "oxidative activity" was inactivated by heating the homogenate for 10 min at 70 °C. Upon addition of a natural epimeric NCC (epiNCC) to the homogenate of senescent or green Sp. wallisii leaves at room temperature, the exogenous epiNCC was oxidized regio- and stereoselectively to 15-OH-epiNCC and 15-OMe-epiNCC. The identical two oxidized epiNCCs were also obtained as products of the oxidation of epiNCC with dicyanodichlorobenzoquinone (DDQ). Water elimination from 15-OH-epiNCC occurred readily and gave a known "yellow" chlorophyll catabolite (YCC). The endogenous oxidation process, described here, may represent the elusive natural path from the colorless NCCs to yellow and pink coloured phyllobilins, which were found in (extracts of) some senescent leaves.

Anti-cancer effects of blue-green alga Spirulina platensis, a natural source of bilirubin-like tetrapyrrolic compounds.

Spirulina platensis is a blue-green alga used as a dietary supplement because of its hypocholesterolemic properties. Among other bioactive substances, it is also rich in tetrapyrrolic compounds closely related to bilirubin molecule, a potent antioxidant and anti-proliferative agent. The aim of our study was to evaluate possible anticancer effects of S. platensis and S. platensis-derived tetrapyrroles using an experimental model of pancreatic cancer. The anti-proliferative effects of S. platensis and its tetrapyrrolic components [phycocyanobilin (PCB) and chlorophyllin, a surrogate molecule for chlorophyll A] were tested on several human pancreatic cancer cell lines and xenotransplanted nude mice. The effects of experimental therapeutics on mitochondrial reactive oxygen species (ROS) production and glutathione redox status were also evaluated. Compared to untreated cells, experimental therapeutics significantly decreased proliferation of human pancreatic cancer cell lines in vitro in a dose-dependent manner (from 0.16 g•L-1 [S. platensis], 60 µM [PCB], and 125 µM [chlorophyllin], p<0.05). The anti-proliferative effects of S. platensis were also shown in vivo, where inhibition of pancreatic cancer growth was evidenced since the third day of treatment (p < 0.05). All tested compounds decreased generation of mitochondrial ROS and glutathione redox status (p = 0.0006; 0.016; and 0.006 for S. platensis, PCB, and chlorophyllin, respectively). In conclusion, S. platensis and its tetrapyrrolic components substantially decreased the proliferation of experimental pancreatic cancer. These data support a chemopreventive role of this edible alga. Furthermore, it seems that dietary supplementation with this alga might enhance systemic pool of tetrapyrroles, known to be higher in subjects with Gilbert syndrome.

Alveolate mitochondrial metabolic evolution: dinoflagellates force reassessment of the role of parasitism as a driver of change in apicomplexans.

Mitochondrial metabolism is central to the supply of ATP and numerous essential metabolites in most eukaryotic cells. Across eukaryotic diversity, however, there is evidence of much adaptation of the function of this organelle according to specific metabolic requirements and/or demands imposed by different environmental niches. This includes substantial loss or retailoring of mitochondrial function in many parasitic groups that occupy potentially nutrient-rich environments in their metazoan hosts. Infrakingdom Alveolata comprises a well-supported alliance of three disparate eukaryotic phyla-dinoflagellates, apicomplexans, and ciliates. These major taxa represent diverse lifestyles of free-living phototrophs, parasites, and predators and offer fertile territory for exploring character evolution in mitochondria. The mitochondria of apicomplexan parasites provide much evidence of loss or change of function from analysis of mitochondrial protein genes. Much less, however, is known of mitochondrial function in their closest relatives, the dinoflagellate algae. In this study, we have developed new models of mitochondrial metabolism in dinoflagellates based on gene predictions and stable isotope labeling experiments. These data show that many changes in mitochondrial gene content previously only known from apicomplexans are found in dinoflagellates also. For example, loss of the pyruvate dehydrogenase complex and changes in tricarboxylic acid (TCA) cycle enzyme complement are shared by both groups and, therefore, represent ancestral character states. Significantly, we show that these changes do not result in loss of typical TCA cycle activity fueled by pyruvate. Thus, dinoflagellate data show that many changes in alveolate mitochondrial metabolism are independent of the major lifestyle changes seen in these lineages and provide a revised view of mitochondria character evolution during evolution of parasitism in apicomplexans.

In vitro DNA-damaging effects of intestinal and related tetrapyrroles in human cancer cells.

Epidemiological studies report a negative association between circulating bilirubin concentrations and the risk for cancer and cardiovascular disease. Structurally related tetrapyrroles also possess in vitro anti-genotoxic activity and may prevent mutation prior to malignancy. Furthermore, few data suggest that tetrapyrroles exert anti-carcinogenic effects via induction of cell cycle arrest and apoptosis. To further investigate whether tetrapyrroles provoke DNA-damage in human cancer cells, they were tested in the single cell gel electrophoresis assay (SCGE). Eight tetrapyrroles (unconjugated bilirubin, bilirubin ditaurate, biliverdin, biliverdin-/bilirubin dimethyl ester, urobilin, stercobilin and protoporphyrin) were added to cultured Caco2 and HepG2 cells and their effects on comet formation (% tail DNA) were assessed. Flow cytometric assessment (apoptosis/necrosis, cell cycle, intracellular radical species generation) assisted in revealing underlying mechanisms of intracellular action. Cells were incubated with tetrapyrroles at concentrations of 0.5, 5 and 17µM for 24h. Addition of 300µM tertiary-butyl hydroperoxide to cells served as a positive control. Tetrapyrrole incubation mostly resulted in increased DNA-damage (comet formation) in Caco2 and HepG2 cells. Tetrapyrroles that are concentrated within the intestine, including protoporphyrin, urobilin and stercobilin, led to significant comet formation in both cell lines, implicating the compounds in inducing DNA-damage and apoptosis in cancer cells found within organs of the digestive system.

Rapid identification of cyclic tetrapyrrolic photosensitisers for photodynamic therapy using on-line hyphenated LC-PDA-MS coupled with photo-cytotoxicity assay.

INTRODUCTION: Photodynamic therapy is a treatment modality that involves site-directed generation of cytotoxic reactive oxygen species by light-activated photosensitisers. OBJECTIVE: In order to rapidly identify new photosensitisers from natural extracts, we developed a liquid chromatography-photodiode array-mass spectrometry (LC-PDA-MS) method to rapidly identify plant extracts that contain photosensitisers, particularly those possessing a cyclic tetrapyrrole structure. METHOD: Six previously isolated compounds (1-6) were identified in bioactive fractions derived from 15 plant extracts on the basis of their chromatographic retention times, UV-visible profiles, accurate mass and fragmentation patterns. RESULTS: Samples containing uncommon photosensitisers were rapidly identified using this method, and subsequent scale-up isolation efforts led to two new compounds (7 and 8) which were confirmed to be active photosensitisers in a photo-cytotoxicity assay. CONCLUSION: This method serves as a useful tool in prioritising samples that may contain new photosensitisers out of a larger group of photo-cytotoxic natural products extracts.

[Photosensitizing anticancer tetrapyrroles: or how photophysics becomes a mechanism of action]. / Tétrapyrroles anticancéreux photosensibilisants : ou comment la photophysique devient mécanisme d'action.

The macrocyclic tetrapyrrole derivatives used for the treatment of certain solid tumors include porphyrins and their chlorine and bacteriochlorin derivatives. These are highly conjugated, rigid molecules characterized by a strong absorbance in the spectral domain from near ultra-violet to far red (350-750 nm). The combination of tetrapyrroles plus light is called dynamic phototherapy (DPT). This combination transforms the molecule to its triplet form which by deactivation generates free radicals and a singlet oxygen from molecular oxygen, causing tumor destruction. Tetrapyrroles are thus, with psoralens, used for the treatment of psoriasis. They are the only drugs whose mechanism of action results exclusively from their electronic and photophysical spectroscopic characteristics. This class of anticancer agents is usually free of any specific cytotoxic effect. We describe here the current elements linking structure and spectroscopy and observations leading to the design of compounds with strong tumor selectivity and optimal cytotoxic properties.

An Arabidopsis mutant that is resistant to the protoporphyrinogen oxidase inhibitor acifluorfen shows regulatory changes in tetrapyrrole biosynthesis.

Several Arabidopsis mutants of the ecotype Dijon were isolated that show resistance to the herbicide acifluorfen, which inactivates protoporphyrinogen oxidase (PPOX), an enzyme of tetrapyrrole biosynthesis. This enzyme provides protoporphyrin for both Mg chelatase and ferrochelatase at the branchpoint, which leads to chlorophyll and heme, respectively. One of the mutations, aci5-3, displays semidominant inheritance. Heterozygous progeny showed yellow-green leaves, while the homozygous seedlings were white and inviable, but could be rescued by supplementing the medium with sugar. Interestingly, the expression of neither of the two forms of PPOX was altered in the mutant, but the rate of synthesis of 5-aminolevulinate, the precursor of all tetrapyrroles, was drastically reduced. Genetic mapping revealed the mutant locus is closely linked to the ch42 marker, which is itself located in the CHLI-1 gene which codes for one of the three subunits of Mg chelatase. The cs mutant also shows a defect in this gene, and test for allelism with aci5-3 confirmed that the two mutations are allelic. Sequencing of the wild type and aci5-3 alleles of CHLI-1 revealed a single base change (G718A), which results in a D240N substitution in the CHLI-1 protein. In the homozygous aci5-3 mutant no CHLI-1 RNA or protein could be detected. Strikingly, CHLH and CHLI-2 transcripts were also absent. This indicates the existence of a feedback-regulatory mechanism that inactivates the genes encoding certain Mg chelatase subunits. The basis for the semidominant inheritance pattern and the relationship between herbicide resistance and modified gene expression is discussed.

Models of the bis-histidine-coordinated ferricytochromes: Mössbauer and EPR spectroscopic studies of low-spin iron(III) tetrapyrroles of various electronic ground states and axial ligand orientations.

The EPR and magnetic Mössbauer spectra of a series of axial ligand complexes of tetrakis(2,6-dimethoxyphenyl)porphyrinatoiron(III), [(2,6-(OMe)(2))(4)TPPFeL(2)](+), where L= N-methylimidazole, 2-methylimidazole, or 4-(dimethylamino)pyridine, of one axial ligand complex of tetraphenylporphyrin, the bis(4-cyanopyridine) complex [TPPFe(4-CNPy)(2)](+), and of one axial ligand complex of tetraphenylchlorin, [TPCFe(ImH)(2)](+), where ImH=imidazole, have been investigated and compared to those of low-spin Fe(III) porphyrinates and ferriheme proteins reported in the literature. On the basis of this and previous complementary spectroscopic investigations, three types of complexes have been identified: those having (d(xy))(2)(d(xz),d(yz))(3) electronic ground states with axial ligands aligned in perpendicular planes (Type I), those having (d(xy))(2)(d(xz),d(yz))(3) electronic ground states with axial ligands aligned in parallel planes (Type II), and those having the novel (d(xz),d(yz))(4)(d(xy))(1) electronic ground state (Type III). A subset of the latter type, with planar axial ligands aligned parallel to each other or strong macrocycle asymmetry that yield rhombic EPR spectra, cannot be created using the porphyrinate ligand. Type I centers are characterized by "large g(max)" EPR spectra with g>3.2 and well-resolved, widely spread magnetic Mössbauer spectra having A(zz)/ g(N)mu(N)>680 kG, with A(xx) negative in sign but much smaller in magnitude than A(zz), while Type II centers have well-resolved rhombic EPR spectra with g(zz)=2.4-3.1 and also less-resolved magnetic Mössbauer spectra, and usually have A(zz)/ g(Nmu(N) in the range of 440-660 kG (but in certain cases as small as 180 kG) and A(xx) again negative in sign but only somewhat smaller (but occasionally larger in magnitude) than A(zz), and Type III centers have axial EPR spectra with g( upper left and right quadrants ) approximately 2.6 or smaller and g( vertical line )<1.0-1.95, but often not resolved, and less-resolved magnetic Mössbauer spectra having A(zz)/ g(N)mu(N) in the range of 270-400 kG, and A(xx) again negative in sign but much smaller in magnitude than A(zz). An exception to this rule is [TPPFe(4-CNPy)(2)](+), which has A(xx)/ g(N)mu(N)=-565 kG, A(yy)/ g(N)mu(N)=629 kG, and A(zz)/ g(N)mu(N)=4 kG. A subset of Type II complexes (Type II') have rhombicities ( V/Delta) much greater than 0.67 and A(zz)/ g(N)mu(N) ranging from 320 to 170 kG, with A(xx) also negative but with the magnitude of A(xx) significantly larger than that of A(zz). These classifications are also observed for a variety of ferriheme proteins, and they lead to linear correlations between A(zz) and either A(xx), g(zz), or V/Delta for Types I and II (but not for A(zz) versus V/Delta for Type II'). Not enough data are yet available on Type III complexes to determine what, if any, correlations may be observed.

Detection of protoporphyrin IX in envelope membranes of pea chloroplasts.

Envelope membranes were prepared from mature pea chloroplasts. The tetrapyrrole contents of envelope membranes were analysed. The envelope membranes of pea chloroplasts contained substantial amounts of protoporphyrin IX and trace amounts of Mg-protoporphyrin IX and its monoester in addition to protochlorophyllide. The protoporphyrin IX content of envelope membranes was 89.25 pmol (mg protein)(-1). Its content in pea envelope membrane was higher than that of protochlorophyllide. The proportion of monovinyl and divinyl forms of protochlorophyllide present in pea chloroplast envelope membrane was 3:7. The significance of the presence of protoporphyrin IX in the envelope membrane is discussed in relation to plastidic Chl biosynthesis.

Taxonomic revision of water-bloom-forming species of oscillatorioid cyanobacteria.

A polyphasic approach was used to clarify the taxonomy of the water-bloom-forming oscillatorioid cyanobacteria. Seventy-five strains of oscillatorioid cyanobacteria were characterized by 16S rDNA sequence analysis, DNA base composition, DNA-DNA hybridization, fatty acid composition, phycobilin pigment composition, complementary chromatic adaptation, morphological characters, growth temperature and salinity tolerance. Phylogenetic analysis based on 16S rDNA sequences divided the strains into six groups, all of which were clearly separated from the type species of the genus Oscillatoria, Oscillatoria princeps Gomont NIVA CYA 150. Therefore, these strains should be classified into genera other than Oscillatoria. Groups I-III were closely related to one another and groups IV-VI were distinct from one another and from groups I to III. Group I was further divided into two subgroups, group I-pc, which includes strains containing only phycocyanin (PC), and group I-pe, which includes strains containing large amounts of phycoerythrin (PE) in addition to PC. This phenotypic distinction was supported by DNA-DNA hybridization studies. Based on the properties examined herein and data from traditional, botanical taxonomic studies, the groups and subgroups were classified into single species and we propose either emended or new taxonomic descriptions for Planktothrix agardhii (type strain NIES 204T), Planktothrix rubescens (type strain CCAP 1459/22T), Planktothrix pseudagardhii sp. nov. (type strain T1-8-4T), Planktothrix mougeotii (type strain TR1-5T), Planktothricoides raciborskii gen. nov., comb. nov. (type strain NIES 207T), Tychonema bourrellyi (type strain CCAP 1459/11BT) and Limnothrix redekei (type strain NIVA CYA 277/1T).

Recombinant type A and B phytochromes from potato. Transient absorption spectroscopy.

The cDNAs encoding full-length type A and B phytochromes (phyA and phyB, respectively) from potato were expressed in inducible yeast systems (Saccharomyces cerevisiae and Pichia pastoris). In addition, a deletion mutant of phyB (delta 1-74) was expressed. The apoproteins were reconstituted into chromoproteins by incorporation of the native chromophore, phytochromobilin (P phi B), and of phycocyanobilin (PCB). The incorporation of P phi B yielded chromoproteins with difference absorptions lambda max at 660 and 712 nm (Pr and Pfr, respectively) for phyA, and at 665 and 723 nm for phyB. All difference maxima of PCB phytochromes are blue-shifted by several nanometers with respect to those obtained with the P phi B chromophore. The deletion construct with PCB shows difference absorption maxima at 652 and 705 nm with the Pfr absorbance considerably reduced. Time-resolved kinetic analysis of a phyB-type phytochrome by nanosecond flash photolysis was performed for the first time. Recombinant full-length phyB afforded transient absorbance changes similar (but not identical) to those of phyA from Avena, whereas the kinetic behavior of these intermediates was very different. Contrary to phyA from Avena, the I700 intermediate from phyB reconstituted with either PCB or P phi B decayed following single exponential kinetics with a lifetime of 87 or 84 microseconds, respectively, at 10 degrees C. The formation of Pfr of PCB-containing recombinant phyB (phyB-PCB) could be fitted with three lifetimes of 9, 127, and 728 ms. The corresponding lifetimes of phyB-P phi B are 22.5, 343, and 2083 ms. Whereas for phyB-PCB all three millisecond lifetimes are related to the formation of Pfr, the 2 s component of phyB-P phi B is concomitant with a rapid recovery of Pr. For recombinant potato phyA and delta 1-74 phyB, no time-resolved data could be obtained due to the limited quantities available. As described for phytochromes of other dicotelydons, the Pfr forms of full-length phyA and PhyB of potato underwent rapid dark conversion to Pr.

Effects of glucose, tetrapyrroles and protein kinase C activators on cell proliferation in cultures of Saccharomyces cerevisiae.

Saccharomyces cerevisiae was inoculated into a yeast nitrogen base with either glycerol or glucose as carbon source. Cell proliferation was followed by colony counts on agar medium. Cells in the glycerol-supplemented medium divided less than once in 10 days. When glucose, 6-deoxy-glucose or protoporphyrin IX was added, the cells had doubling times of about 24 h and increased in number to about 0.5 x 10(6) cells ml-1. Addition of either of the protein kinase C activators oleoyl-acetyl-glycerol or phorbol-12-myristate-13-acetate did not activate cell proliferation in the glycerol medium. However, when (i) glucose was combined with either protoporphyrin IX or chlorophyllin, or (ii) either protoporphyrin IX or chlorophyllin was combined with either of the protein kinase C activators, the cells had doubling times of about 12 h. Hence, (i) glucose can act as both a carbon source and a signalling molecule for proliferation, and (ii) two systems are involved in activating cell proliferation in S. cerevisiae: one operating through a protein kinase C system and another through a guanylate cyclase system.

Cloning and characterisation of genes for tetrapyrrole biosynthesis from the cyanobacterium Anacystis nidulans R2.

The genes for 5-aminolevulinic acid dehydratase (ALAD) and uroporphyrinogen III synthase (UROS), two enzymes in the biosynthetic pathway for tetrapyrroles, were independently isolated from a plasmid-based genomic library of Anacystis nidulans R2 (also called Synechococcus sp. PCC7942), by their ability to complement Escherichia coli strains carrying mutations in the equivalent genes (hemB and hemD respectively). The identity of the genes was confirmed by comparing the appropriate enzyme activities in complemented and mutant strains. Subclones of the original plasmids that were also capable of complementing the mutants were sequenced. The inferred amino acid sequence of the cyanobacterial HemB protein indicates a significant difference in the metal cofactor requirement from the higher-plant enzymes, which was confirmed by overexpression and biochemical analysis. The organisation of the cyanobacterial hemD locus differs markedly from other prokaryotes. Two open reading frames were found immediately upstream of hemD. The product of one shows considerable similarity to published sequences from other organisms for uroporphyrinogen III methylase (UROM), an enzyme involved in the production of sirohaem and cobalamins (including vitamin B-12). The product of the other shows motifs which are similar to those found in proteins responsible for metabolic regulation in yeast and indicates that this family of transcription control proteins, which has previously been reported only from eukaryotes, is also represented in prokaryotes.

Mutational analysis of the pea phytochrome A chromophore pocket: chromophore assembly with apophytochrome A and photoreversibility.

Ten site-specific mutants of pea apophytochrome A were expressed in Saccharomyces cerevisiae and analyzed for chromophore assembly with apoprotein and photoreversible absorbance changes. The mutants constitute two specific changes for each of five conserved amino acid residues located in the microenvironment of the chromophore attachment residue, which is Cys-323 in pea phytochrome A. All mutant apophytochromes were autocatalytically able to covalently attach phycocyanobilin, indicating that there were no major structural perturbations in the apoproteins. However, the rate of chromophore ligation varied significantly among the mutants. Spectrally, the mutant holophytochromes are of three types: mutant phytochromes that are indistinguishable from the wild-type adduct, mutants with blue-shifted Pr and Pfr absorption maxima compared to the wild-type adduct, and mutants that are not photoreversible. From an analysis of the results, we concluded that the residues Asp-309, Arg-318, His-321, and Gln-326 are probably not catalytically involved in the chromophore ligation reaction, but some residues may play significant structural and stereochemical roles. Arg-318 might anchor the chromophore, as has been suggested [Partis, M. D., & Grimm, R. (1990) Z. Naturforsch, 45c, 987-998; Parker, W., et al. (1993) Bioconjugate Chem. (in press)]. The conserved Gln-326, three residues downstream from the chromophore attachment site, is not electrostatically critical for the spectral integrity and photoreversibility of phytochrome, but this residue is sterically important to the lyase activity. It appears that the role of the five amino acid residues in the N- and C-terminal vicinities of the chromophore binding Cys-323 is structural rather than catalytic for the ligation reaction.

Investigation of the subcellular location of the tetrapyrrole-biosynthesis enzyme coproporphyrinogen oxidase in higher plants.

The subcellular location of two enzymes in the biosynthetic pathway for protoporphyrin IX, coproporphyrinogen (coprogen) oxidase (EC 1.3.3.3) and protoporphyrinogen (protogen) oxidase (EC 1.3.3.4) has been investigated in etiolated pea (Pisum sativum) leaves and spadices of cuckoo-pint (Arum maculatum). Plant tissue homogenized in isotonic buffer was subjected to subcellular fractionation to prepare mitochondria and plastids essentially free of contamination by other cellular organelles, as determined by marker enzymes. Protogen oxidase activity measured fluorimetrically was reproducibly found in both mitochondria and etioplasts. In contrast, coprogen oxidase could be detected only in etioplasts, using either a coupled fluorimetric assay or a sensitive radiochemical method. The implications of these results for the synthesis of mitochondrial haem in plants is discussed.

In vitro assembly of apophytochrome and apophytochrome deletion mutants expressed in yeast with phycocyanobilin.

Recombinant pea type I phytochrome apoprotein expressed in yeast is shown to assemble in vitro with phycocyanobilin to produce a photoreversible phytochrome-like adduct. As an initial investigation of the amino acid sequence requirements for chromophore incorporation, three phyA gene product deletion mutants were produced in yeast. Truncation of the N-terminal tail to residue 46 demonstrates that this region is not critical to bilin attachment, but a deletion mutant lacking 222 amino acids from the N terminus failed to yield holophytochrome in vitro, under the same conditions. A mutant comprising a deletion of the C terminus to residue 548 showed bilin incorporation and red/far-red photoreversibility, indicating that bilin-apophytochrome assembly still occurred even when the entire C-terminal domain was truncated.