Chlorophyllides: Preparation, Purification, and Application.

Chlorophyllides can be found in photosynthetic organisms. Generally, chlorophyllides have a-, b-, c-, d-, and f-type derivatives, and all chlorophyllides have a tetrapyrrole structure with a Mg ion at the center and a fifth isocyclic pentanone. Chlorophyllide a can be synthesized from protochlorophyllide a, divinyl chlorophyllide a, or chlorophyll. In addition, chlorophyllide a can be transformed into chlorophyllide b, chlorophyllide d, or chlorophyllide f. Chlorophyllide c can be synthesized from protochlorophyllide a or divinyl protochlorophyllide a. Chlorophyllides have been extensively used in food, medicine, and pharmaceutical applications. Furthermore, chlorophyllides exhibit many biological activities, such as anti-growth, antimicrobial, antiviral, antipathogenic, and antiproliferative activity. The photosensitivity of chlorophyllides that is applied in mercury electrodes and sensors were discussed. This article is the first detailed review dedicated specifically to chlorophyllides. Thus, this review aims to describe the definition of chlorophyllides, biosynthetic routes of chlorophyllides, purification of chlorophyllides, and applications of chlorophyllides.

Facile production of chlorophyllides using recombinant CrCLH1 and their cytotoxicity towards multidrug resistant breast cancer cell lines.

The purity of chlorophylls plays one of the key role for the production of chlorophyllides. We have designed a facile method for chlorophyll purification by twice solvent extraction. Twice extraction causes the loss of chlorophylls, but the purity of total chlorophylls can be enhanced 182%. Then, the purified chlorophylls can be converted to relatively pure chlorophyllides facilely. The results show that higher purity of chlorophyllides could be obtained when purified chlorophylls (ethanol-hexane extract) was used as starting materials than that of crude chlorophylls (ethanol-only extract). In biocompatibility test, the results showed that the prepared chlorophyllides can be applied as biomaterials. When the prepared chlorophyllides were applied to anticancer tests, they were active both in MCF7 and MDA-MB-231 (multidrug resistant breast cancer cells) cell lines. In addition, the results suggested that the prepared chlorophyllides could be a potential candidate of combination therapy with doxorubicin to breast cancers.

A Review of Bacteriochlorophyllides: Chemical Structures and Applications.

Generally, bacteriochlorophyllides were responsible for the photosynthesis in bacteria. Seven types of bacteriochlorophyllides have been disclosed. Bacteriochlorophyllides a/b/g could be synthesized from divinyl chlorophyllide a. The other bacteriochlorophyllides c/d/e/f could be synthesized from chlorophyllide a. The chemical structure and synthetic route of bacteriochlorophyllides were summarized in this review. Furthermore, the potential applications of bacteriochlorophyllides in photosensitizers, immunosensors, influence on bacteriochlorophyll aggregation, dye-sensitized solar cell, heme synthesis and for light energy harvesting simulation were discussed.

Dual role of the active site 'lid' regions of protochlorophyllide oxidoreductase in photocatalysis and plant development.

Protochlorophyllide oxidoreductase (POR) catalyses reduction of protochlorophyllide (Pchlide) to chlorophyllide, a light-dependent reaction of chlorophyll biosynthesis. POR is also important in plant development as it is the main constituent of prolamellar bodies in etioplast membranes. Prolamellar bodies are highly organised, paracrystalline structures comprising aggregated oligomeric structures of POR-Pchlide-NADPH complexes. How these oligomeric structures are formed and the role of Pchlide in oligomerisation remains unclear. POR crystal structures highlight two peptide regions that form a 'lid' to the active site, and undergo conformational change on binding Pchlide. Here, we show that Pchlide binding triggers formation of large oligomers of POR using size exclusion chromatography. A POR 'octamer' has been isolated and its structure investigated by cryo-electron microscopy at 7.7 Å resolution. This structure shows that oligomer formation is most likely driven by the interaction of amino acid residues in the highly conserved lid regions. Computational modelling indicates that Pchlide binding stabilises exposure of hydrophobic surfaces formed by the lid regions, which supports POR dimerisation and ultimately oligomer formation. Studies with variant PORs demonstrate that lid residues are involved in substrate binding and photocatalysis. These highly conserved lid regions therefore have a dual function. The lid residues position Pchlide optimally to enable photocatalysis. Following Pchlide binding, they also enable POR oligomerisation - a process that is reversed through subsequent photocatalysis in the early stages of chloroplast development.

Nitric oxide regulates chlorophyllide biosynthesis and singlet oxygen generation differently between Arabidopsis and barley.

Nitric oxide (NO) has a general inhibitory effects on chlorophyll biosynthesis, especially to the step of 5-aminolevulinic acid (ALA) biosynthesis and protochlorophyllide (Pchlide) to chlorophyllide (Chlide) conversion (responsible by the NADPH:Pchlide oxidoreductase POR). Previous study suggested that barley large POR aggregates may be generated by dithiol oxidation of cysteines of two POR monomers, which can be disconnected by some reducing agents. POR aggregate assembly may be correlated with seedling greening in barley, but not in Arabidopsis. Thus, NO may affect POR activity and seedling greening differently between Arabidopsis and barley. We proved this assumption by non-denaturing gel-analysis and reactive oxygen species (ROS) monitoring during the greening. NO treatments cause S-nitrosylation to POR cysteine residues and disassembly of POR aggregates. This modification reduces POR activity and induces Pchlide accumulation and singlet oxygen generation upon dark-to-high-light shift (and therefore inducing photobleaching lesions) in barley leaf apex, but not in Arabidopsis seedlings. ROS staining and ROS-related-gene expression detection confirmed that superoxide anion and singlet oxygen accumulated in barley etiolated seedlings after the NO treatments, when exposed to a fluctuating light. The data suggest that POR aggregate assembly may be correlated with barley chlorophyll biosynthesis and redox homeostasis during greening. Cysteine S-nitrosylation may be one of the key reasons for the NO-induced inhibition to chlorophyll biosynthetic enzymes.

Chlorophylls and their Derivatives Used in Food Industry and Medicine.

BACKGROUND & OBJECTIVE: Thylakoids and chloroplasts harbor several vital metabolic processes, but are most importantly associated with photosynthesis. The undisturbed functioning of this process necessitates the ceaseless synthesis of photosynthetic pigments, including closed tetrapyrroles such as chlorophylls (Chls). Chls probably represent the most abundant natural pigment molecules which are via photosynthesis not only crucial for the autotrophic production of food sources for heterotrophic organisms but have also contributed to oxygen production essential for aerobic metabolism. This review first briefly discusses the physico-chemical properties, biosynthesis, occurrence, in vivo localization and roles of the different Chl pigments. Then we provide a detailed overview of their potential applications in the food industry and medicine. These include the use of Chls and their derivatives (different chlorophyllins) as food colorants (identified as E140 and E141 in the European Union). METHOD: Different sources used for industrial extraction as well as different factors influencing pigment stability during processing are also critically reviewed. The problems surrounding the nomenclature, the production and the composition of different chlorophyllin mixtures are also discussed. RESULTS & CONCLUSION: Finally, a comprehensive overview of the health benefits and potential medicinal applications of these pigments and the future directions of research in these fields are provided.

Chlorophyll biosynthesis gene evolution indicates photosystem gene duplication, not photosystem merger, at the origin of oxygenic photosynthesis.

An open question regarding the evolution of photosynthesis is how cyanobacteria came to possess the two reaction center (RC) types, Type I reaction center (RCI) and Type II reaction center (RCII). The two main competing theories in the foreground of current thinking on this issue are that either 1) RCI and RCII are related via lineage divergence among anoxygenic photosynthetic bacteria and became merged in cyanobacteria via an event of large-scale lateral gene transfer (also called "fusion" theories) or 2) the two RC types are related via gene duplication in an ancestral, anoxygenic but protocyanobacterial phototroph that possessed both RC types before making the transition to using water as an electron donor. To distinguish between these possibilities, we studied the evolution of the core (bacterio)chlorophyll biosynthetic pathway from protoporphyrin IX (Proto IX) up to (bacterio)chlorophyllide a. The results show no dichotomy of chlorophyll biosynthesis genes into RCI- and RCII-specific chlorophyll biosynthetic clades, thereby excluding models of fusion at the origin of cyanobacteria and supporting the selective-loss hypothesis. By considering the cofactor demands of the pathway and the source genes from which several steps in chlorophyll biosynthesis are derived, we infer that the cell that first synthesized chlorophyll was a cobalamin-dependent, heme-synthesizing, diazotrophic anaerobe.

Methods for analysis of photosynthetic pigments and steady-state levels of intermediates of tetrapyrrole biosynthesis.

Tetrapyrroles and carotenoids are required for many indispensable functions in photosynthesis. Tetrapyrroles are essential metabolites for photosynthesis, redox reaction, and detoxification of reactive oxygen species and xenobiotics, while carotenoids function as accessory pigments, in photoprotection and in attraction to animals. Their branched metabolic pathways of synthesis and degradation are tightly controlled to provide adequate amounts of each metabolite (carotenoids/tetrapyrroles) and to prevent accumulation of photoreactive intermediates (tetrapyrroles). Many Arabidopsis mutants and transgenic plants have been reported to show variations in steady-state levels of tetrapyrrole intermediates and contents of different carotenoid species. It is a challenging task to determine the minute amounts of these metabolites to assess the metabolic flow and the activities of both pigment-synthesising and degrading pathways, to unravel limiting enzymatic steps of these biosynthetic pathways, and to characterise mutants with accumulating intermediates. In this chapter, we present a series of methods to qualify and quantify anabolic and catabolic intermediates of Arabidopsis tetrapyrrole metabolism, and describe a common method for quantification of different plant carotenoid species. Additionally, we introduce two methods for quantification of non-covalently bound haem. The approach of analysing steady-state levels of tetrapyrrole intermediates in plants, when applied in combination with analyses of transcripts, proteins, and enzyme activities, enables the biochemical and genetic elucidation of the tetrapyrrole pathway in wild-type plants, varieties, and mutants. Steady-state levels of tetrapyrrole intermediates are only up to 1/1,000 of the amounts of the accumulating end-products, chlorophyll, and haem. Although present in very low amounts, the accumulation and availability of tetrapyrrole intermediates have major consequences on the physiology and activity of chloroplasts due to their additional photoreactive and possible signalling functions. Although adjusted for Arabidopsis tetrapyrrole metabolites, the presented methods can also be applied for analysis of cyanobacterial and other plant tetrapyrroles.

Chlorophyll biosynthesis in bacteria: the origins of structural and functional diversity.

The use of photochemical reaction centers to convert light energy into chemical energy, chlorophototrophy, occurs in organisms belonging to only five eubacterial phyla: Cyanobacteria, Proteobacteria, Chlorobi, Chloroflexi, and Firmicutes. All chlorophototrophs synthesize two types of pigments: (a) chlorophylls and bacteriochlorophylls, which function in both light harvesting and uniquely in photochemistry; and (b) carotenoids, which function primarily as photoprotective pigments but can also participate in light harvesting. Although hundreds of carotenoids have been identified, only 12 types of chlorophylls (Chl a, b, d; divinyl-Chl a and b; and 8(1)-hydroxy-Chl a) and bacteriochlorophylls (BChl a, b, c, d, e, and g) are currently known to occur in bacteria. This review summarizes recent progress in the identification of genes and enzymes in the biosynthetic pathways leading to Chls and BChls, the essential tetrapyrrole cofactors of photosynthesis, and addresses the mechanisms for generating functional diversity for solar energy capture and conversion in chlorophototrophs.

Two routes of chlorophyllide synthesis that are differentially regulated by light in barley (Hordeum vulgare L.).

NADPH-protochlorophyllide oxidoreductase (POR; EC 1.6.99.1) catalyzes the only known light-dependent step in chlorophyll synthesis of higher plants, the reduction of protochlorophyllide (Pchlide) to chlorophyllide. In barley, two distinct immunoreactive POR proteins were identified. In contrast to the light-sensitive POR enzyme studied thus far (POR-A), levels of the second POR protein remained constant in seedlings during the transition from dark growth to the light and in green plants. The existence of a second POR-related protein was verified by isolating and sequencing cDNAs that encode a second POR polypeptide (POR-B) with an amino acid sequence identity of 75% to the POR-A. In the presence of NADPH and Pchlide, the in vitro-synthesized POR-A and POR-B proteins could be reconstituted to ternary enzymatically active complexes that reduced Pchlide to chlorophyllide only after illumination. Even though the in vitro activities of the two enzymes were similar, the expression of their genes during the light-induced transformation of etiolated to green seedlings was distinct. While the POR-A mRNA rapidly declined during illumination of dark-grown seedlings and soon disappeared, POR-B mRNA remained at an approximately constant level in dark-grown and green seedlings. Thus these results suggest that chlorophyll synthesis is controlled by two light-dependent POR enzymes, one that is active only transiently in etiolated seedlings at the beginning of illumination and the other that also operates in green plants.

Chloroplast biogenesis: quantitative determination of monovinyl and divinyl chlorophyll(ide) a and b by spectrofluorometry.

Simultaneous equations for the determination of monovinyl (MV) and divinyl (DV) Chl a and b by spectrofluorometry in unsegregated mixtures of these tetrapyrroles were derived. The same equations can also be used for the quantitative determination of MV and DV chlorophyllide (Chlide) a and b in unpurified mixtures, after extraction of the Chls in hexane. The equations used differences in the Soret excitation maxima of these tetrapyrroles in ether at 77 degrees K, in order to correct the Soret excitation overlap between MV and DV Chl(ide) a and between MV and DV Chl(ide) b.

Biosynthesis of a chlorophyllide b-like pigment in phenanthroline-treated Chlamydomonas reinhardtii y-1.

Incubation of degreened Chlamydomonas reinhardtii y-1 cells in the dark with m-phenanthroline induced de novo synthesis of a chlorophyllide b-like pigment. The rate of synthesis of this pigment in the dark was greater than that of total chlorophyll in illuminated cells. Most of the newly synthesized pigment was excreted into the culture medium. The product was extracted from the medium as the metal-free pheophorbide, which had a fluorescence excitation maximum at 428 +/- 1 nm and an emission maximum at 657 +/- 1 nm (E428F657) in ethyl acetate (E427F657 in diethyl ether). Three pheophorbide species were extracted from the medium of green cells treated in the dark, a minor component with a spectrum (E410F670) identical to demetallated chlorophyll a, and two major species with spectral values of E428F657 and E433F657. The latter, predominant form had a spectrum identical to demetallated chlorophyll b, which was purified from the algal cells. E428F657 and E433F657 reacted with hydroxylamine and Girard's T-reagent, which caused a shift in the fluorescence emission maximum to 668 nm. Pheophytin b, which contains an aldehyde group, exhibited an identical spectral shift when treated in the same way, but pheophytin a or porphyrin biosynthetic intermediates did not. Proton NMR analysis of the E428F657 chlorin produced by yellow cells treated with m-phenanthroline confirmed the presence of an aldehydic proton. Chelating and nonchelating phenanthroline analogs equally stimulated synthesis of this product.

Chloroplast biogenesis: biosynthesis of protochlorophyll(ide) via acidic and fully esterified biosynthetic branches in higher plants.

The operation of two parallel biosynthetic pathways during the biosynthesis of the protochlorophyll(ide) pool of cucumber cotyledons was probed with the use of several [14C]tetrapyrroles in vitro. A comparison of the ratio (R) of delta-amino[14C]levulinic acid incorporation into protochlorophyllide/14C incorporation into protochlorophyllide ester with other incorporation ratios, RX, where X represents any of several [14C]tetrapyrrole substrates, allowed us to determine which exogenous 14C-labeled substrate was common precursor of protochlorophyllide and protochlorophyllide ester and which one was not. From such comparisons, a biosynthetic pathway of protochlorophyll(ide) biosynthesis is inferred. According to this pathway, protochlorophyllide is formed via an acidic (mono/dicarboxylic) biosynthetic branch, while protochlorophyllide ester is formed via a fully esterified (neutral) biosynthetic branch. The two biosynthetic branches are linked together by porphyrin ester synthetases at the level of protochlorophyllides and possibly at the level of the photoporphyrin IX and the magnesium protoporphyrin monoester pools.

Reconstitution of chlorophyllide formation by isolated etioplast membranes.

1. The reconstitution of chlorophyllide biosynthesis by barley etioplast membranes is described. 2. The process is dependent on the additon of NADPH and protochlorophyllide and on illumination, which can be either continuous or intermittent. 3. The reconstituted process involves spectroscopically similar intermediates to the native reaction in whole leaves. 4. Steps in the process are an initial enzymic formation in the dark of a photoactive complex, P638/652 (probably a ternary protochlorophyllide-NADPH-enzyme complex), followed by a very rapid light-dependent hydrogen transfer from the NADPH to the protochlorophyllide giving chlorophyllide giving chlorophyllide, finally releasing the enzyme for repeating the process. 5. A continuous assay for the system regenerating complex P638/652 was devised on the basis of monitoring chlorophyllide formation. 6. The pH optimum of the reaction is at 6.9 and Km values for protochlorophyllide and NADPH are 0.46 and 35 micron respectively. 7. The reaction is associated specifically with the etioplast membrane fraction. 8. Activities of the system assayed in vitro are more than adequate to account for rates of chlorophyll formation in vivo.