Revealing the significance of chlorophyll b in the moss Physcomitrium patens by knocking out two functional chlorophyllide a oxygenase.

The chlorophyllide a oxygenase (CAO) plays a crucial role in the biosynthesis of chlorophyll b (Chl b). In the moss Physcomitrium patens (P. patens), two distinct gene copies, PpCAO1 and PpCAO2, are present. In this study, we investigate the differential expression of these CAOs following light exposure after a period of darkness (24 h) and demonstrate that the accumulation of Chl b is only abolished when both genes are knocked out. In the ppcao1cao2 mutant, most of the antenna proteins associated with both photosystems (PS) I and II are absent. Despite of the existence of LHCSR proteins and zeaxanthin, the mutant exhibits minimal non-photochemical quenching (NPQ) capacity. Nevertheless, the ppcao1cao2 mutant retains a certain level of pseudo-cyclic electron transport to provide photoprotection for PSI. These findings shed light on the dual dependency of Chl b synthesis on two CAOs and highlight the distinct effects of Chl b deprival on PSI and PSII core complexes in P. patens, a model species for bryophytes.

Evidence of increases of phytol and chlorophyllide by enzymatic dephytylation of chlorophylls in smoothie made from spinach leaves.

Phytol is a diterpene alcohol found abundantly in nature as the phytyl side chain of chlorophylls. Free form of phytol and its metabolites have been attracting attention because they have a potential to improve the lipid and glucose metabolism. On the other hand, phytol is unfavorable for those who suffering from Refsum's disease. However, there is little information on the phytol contents in leafy vegetables rich in chlorophylls. This study indicated that raw spinach leaves contain phytol of 0.4-1.5 mg/100 g fresh weight. Furthermore, crude enzyme extracted from the leaves showed the enzyme activities involved in dephytylation of chlorophyll derivatives and they were high at mild alkaline pH and around 45°C, and lowered at 55°C or above. Under the optimum pH and temperature for such enzymes determined in the model reaction using the crude enzyme, phytol content in the smoothie made from raw spinach leaves increased with an increase of chlorophyllide, another reaction product. Comparison between the increased amounts of phytol and chlorophyllide showed that the enzymatic dephytylation of chlorophylls was critically responsible for the increase of phytol in the smoothie. PRACTICAL APPLICATION: Phytol, which is released by the enzymes related to chlorophyll metabolism in plants, has been investigated because of its potential abilities to improve the lipid metabolism and blood glucose level. In contrast to such health benefits, they are known to be toxic for patients suffering from Refsum's disease. This research for the first time reports the phytol content in raw spinach leaves and that phytol can be increased in the smoothie made from spinach leaves by the action of endogenous enzymes on chlorophyll derivatives under a certain condition. These results help control phytol content in the smoothies.

Structural Characterization of the Chlorophyllide a Oxygenase (CAO) Enzyme Through an In Silico Approach.

Chlorophyllide a oxygenase (CAO) is responsible for converting chlorophyll a to chlorophyll b in a two-step oxygenation reaction. CAO belongs to the family of Rieske-mononuclear iron oxygenases. Although the structure and reaction mechanism of other Rieske monooxygenases have been described, a member of plant Rieske non-heme iron-dependent monooxygenase has not been structurally characterized. The enzymes in this family usually form a trimeric structure and electrons are transferred between the non-heme iron site and the Rieske center of the adjoining subunits. CAO is supposed to form a similar structural arrangement. However, in Mamiellales such as Micromonas and Ostreococcus, CAO is encoded by two genes where non-heme iron site and Rieske cluster localize on the distinct polypeptides. It is not clear if they can form a similar structural organization to achieve the enzymatic activity. In this study, the tertiary structures of CAO from the model plant Arabidopsis thaliana and the Prasinophyte Micromonas pusilla were predicted by deep learning-based methods, followed by energy minimization and subsequent stereochemical quality assessment of the predicted models. Furthermore, the chlorophyll a binding cavity and the interaction of ferredoxin, which is the electron donor, on the surface of Micromonas CAO were predicted. The electron transfer pathway was predicted in Micromonas CAO and the overall structure of the CAO active site was conserved even though it forms a heterodimeric complex. The structures presented in this study will serve as a basis for understanding the reaction mechanism and regulation of the plant monooxygenase family to which CAO belongs.

Chlorophyll dephytylase 1 and chlorophyll synthase: a chlorophyll salvage pathway for the turnover of photosystems I and II.

Chlorophyll (Chl) is made up of the tetrapyrrole chlorophyllide and phytol, a diterpenoid alcohol. The photosynthetic protein complexes utilize Chl for light harvesting to produce biochemical energy for plant development. However, excess light and adverse environmental conditions facilitate generation of reactive oxygen species, which damage photosystems I and II (PSI and PSII) and induce their turnover. During this process, Chl is released, and is thought to be recycled via dephytylation and rephytylation. We previously demonstrated that Chl recycling in Arabidopsis under heat stress is mediated by the enzymes chlorophyll dephytylase 1 (CLD1) and chlorophyll synthase (CHLG) using chlg and cld1 mutants. Here, we show that the mutants with high CLD1/CHLG ratio, by different combinations of chlg-1 (a knock-down mutant) and the hyperactive cld1-1 alleles, develop necrotic leaves when grown under long- and short-day, but not continuous light conditions, owing to the accumulation of chlorophyllide in the dark. Combination of chlg-1 with cld1-4 (a knock-out mutant) leads to reduced chlorophyllide accumulation and necrosis. The operation of CLD1 and CHLG as a Chl salvage pathway was also explored in the context of Chl recycling during the turnover of Chl-binding proteins of the two photosystems. CLD1 was found to interact with CHLG and the light-harvesting complex-like proteins OHP1 and LIL3, implying that auxiliary factors are required for this process.

Ferroptosis triggered by dihydroartemisinin facilitates chlorin e6 induced photodynamic therapy against lung cancerthrough inhibiting GPX4 and enhancing ROS.

Photodynamic therapy (PDT) is noninvasive, low toxicity, and photo-selective, but may be resisted by malignant cells. A previous study found chlorin e6 (Ce6) mediated PDT showed drug resistance in lung cancer cells (LLC), which may be associated with PDT-induced DNA damage response (DDR). DDR may up-regulate glutathione peroxidase 4 (GPX4), which in turn degrade ROS induced by PDT. However, dihydroartemisinin (DHA) was found to down-regulate GPX4. Accordingly, the DHA was hypothesized to improve the resistance to PDT. The present work explores the mechanism of Ce6 mediated drug resistance and reveals whether DHA can enhance the efficacy of PDT by suppressing GPX4. The in vitro experiments found Ce6 treatment did not inhibit the viability of LLC within 6 h without inducing significant apoptosis, suggesting LLC were resistant to PDT. Further investigation demonstrated PDT could damage DNA and up-regulate GPX4, thus degrading the generated ROS. DHA effectively inhibited the viability of LLC and induced apoptosis. Importantly, DHA displayed a prominent inhibitory effect on the GPX4 expression and thereby triggered ferroptosis. Combining DHA with Ce6 for treatment of LLC resulted in the suppressed GPX4 and elevated ROS. Finally, the findings showed DHA combined with Ce6 exhibited superb anti-lung cancer efficacy. In summary, Ce6 PDT damages DNA, up-regulates GPX4 to degrade ROS, thereby inducing drug resistance. Down-regulation of GPX4 by DHA-triggered ferroptosis significantly enhances the efficacy of PDT. This study provides an outstanding theoretical basis for the regulation of the intratumoral redox system and improving PDT efficacy against lung cancer by herbal monomer DHA.

Hitchhiking Nanoparticles: Mesenchymal Stem Cell-Mediated Delivery of Theranostic Nanoparticles.

Nanotechnology has emerged as a promising solution to permanent elimination of cancer. However, nanoparticles themselves lack specificity to tumors. Due to enhanced migration to tumors, mesenchymal stem cells (MSCs) were suggested as cell-mediated delivery vehicles of nanoparticles. In this study, we have constructed a complex composed of photoluminescent quantum dots (QDs) and a photosensitizer chlorin e6 (Ce6) to obtain multifunctional nanoparticles, combining cancer diagnostic and therapeutic properties. QDs serve as energy donors-excited QDs transfer energy to the attached Ce6 via Förster resonance energy transfer, which in turn generates reactive oxygen species. Here, the physicochemical properties of the QD-Ce6 complex and singlet oxygen generation were measured, and the stability in protein-rich media was evaluated, showing that the complex remains the most stable in protein-free medium. In vitro studies on MSC and cancer cell response to the QD-Ce6 complex revealed the complex-loaded MSCs' potential to transport theranostic nanoparticles and induce cancer cell death. In vivo studies proved the therapeutic efficacy, as the survival of tumor-bearing mice was statistically significantly increased, while tumor progression and metastases were slowed down.

Macrophages mediated delivery of chlorin e6 and treatment of lung cancer by photodynamic reprogramming.

Photodynamic therapy (PDT) is an emerging anti-tumor strategy.Photosensitizer chlorin e6 (Ce6) can induce photodynamic effect to selectively damage lung cancer cells.In order to further improve its tumor targeting ability, macrophages can be applied as carrier to deliver Ce6 to lung cancer.Tumor associated macrophages (TAM) are important immunocytes in lung cancer immune microenvironment. TAM play crucial role in tumor promotion due to the Immunosuppressive property, reprogramming phenotype of TAM therefore has become a promising strategy.Based on this, in the present study, we suppose that TAM can be used as carrier to deliver Ce6 to lung cancer and be reprogrammed to M1 phenotype by photodynamic action to mediate anti-lung cancer efficacy.The results showed TAM could load with Ce6 and keep viability in the absence of near infrared irradiation (NIR).Moreover, Its viability decreased little within 10 h after NIR.Ce6-loaded TAM could deliver Ce6 to lung cancer cells and retain some drugs in TAM per se.After NIR, phagocytosis of macrophages was enhanced. The expressions of GBP5, iNOS and MHC-II was up-regulated, which indicated TAM were polarized to M1 phenotype.Finally, the study also found the reprogrammed macrophages could inhibit the proliferation and promote the apoptosis of lung cancer cells.These results suggested that macrophages could deliver Ce6 to lung cancer and exhibit anti-lung cancer effect through photodynamic reprogramming.This study provides a novel approach for combining photodynamic action with anti-tumor immunotherapy.

Exosomes synergized with PIONs@E6 enhance their immunity against hepatocellular carcinoma via promoting M1 macrophages polarization.

BACKGROUND: Hepatocellular carcinoma (HCC) is easy to relapse after resection for its lack of anti-tumor immunity due to pro-tumorigenesis by promoting M2 type macrophage polarization. Recent studies have shown that exosomes are closely related to the occurrence and development of HCC. Antigenic exosomes from HCC are able to polarize into alternatively activated macrophages M2, but do not stimulate M1 macrophages polarization. Iron oxide nanoparticles (IONs) have been demonstrated to be able to promote M1 macrophages polarization. This research was to explore exosomes as vehicles to synergize with pegylated IONs loaded with chlorin e6 (PIONs@E6) to enhance their immunity against HCC via promoting M1 macrophages polarization. MATERIALS AND METHODS: PIONs@E6 was synthesized and then characterized by chemico-physical analysis, transmission electron microscope (TEM), respectively. After characterization of PIONs-contained exosomes by TEM, and then the exosomal surface specific molecules CD9 and CD63 were determined by Western Blotting assay. Markers of M1 macrophage polarization in vitro and in vivo were analyzed by enzyme linked immunosorbent assay (ELISA) and flow cytometry, respectively. Intracellular reactive oxygen species (ROS) in macrophages were analyzed using a Spectra Max fluorescence microplate reader. Inhibitory effect of PIONs-contained exosomes on HCC was evaluated by monitoring tumor growth in an in vivo xenograft mice model. RESULTS: PIONs@E6 showed good water solubility with a core diameter around 10 nm and a hydrate diameter around 37 nm. The expression of exosome specific markers CD9 and CD63 was kept at a high level. PIONs-contained exosomes can dose-dependently promote M1 macrophages polarization in vitro and in vivo. Of note, PIONs-contained exosomes could initiate a significantly higher level of ROS in macrophages and remarkably inhibit the tumor growth in mice bearing HCC xenograft. CONCLUSION: Exosomes as vehicles could be synergized with PIONs@E6 to enhance their immunity against HCC via promoting M1 macrophages polarization.

Characterization of Seedling Greening Process in Plant Photomorphogenesis.

Seedling deetiolation is a hallmark of the photomorphogenic response, and successful conversion of protochlorophyllide (Pchlide) into chlorophyllide during initial light exposure is critical for plant survival and growth. Here we describe the seedling deetiolation process of two typical mutants pif3 and flu by analysis of the cotyledons greening, Pchlide content, and reactive oxygen species (ROS) production and summarize a set of general methods for the research of seedling greening.

Detection of 132-carboxy-chlorin produced by the in vitro BciC enzymatic hydrolysis of zinc chlorophyllide.

Green photosynthetic bacteria with an efficient light-harvesting system contain special chlorophyll molecules, called bacteriochlorophylls c, d, e, in their main antennae. In the biosynthetic pathway, a BciC enzyme is proposed to catalyze the hydrolysis of the C132-methoxycarbonyl group of chlorophyllide a, but the resulting C132-carboxy group has not been detected yet because it is spontaneously removed due to the instability of the ß-keto-carboxylic acid. In this study, the in vitro BciC enzymatic reactions of zinc methyl (131R/S)-hydroxy-mesochlorophyllides a were examined and a carboxylic acid possessing the C132S-OH was first observed as the hydrolyzed product of the C132-COOCH3.

Compensation Mechanism of the Photosynthetic Apparatus in Arabidopsis thaliana ch1 Mutants.

The origin of chlorophyll b deficiency is a mutation (ch1) in chlorophyllide a oxygenase (CAO), the enzyme responsible for Chl b synthesis. Regulation of Chl b synthesis is essential for understanding the mechanism of plant acclimation to various conditions. Therefore, the main aim of this study was to find the strategy in plants for compensation of low chlorophyll content by characterizing and comparing the performance and spectral properties of the photosynthetic apparatus related to the lipid and protein composition in four selected Arabidopsis ch1 mutants and two Arabidopsis ecotypes. Mutation in different loci of the CAO gene, viz., NW41, ch1.1, ch1.2 and ch1.3, manifested itself in a distinct chlorina phenotype, pigment and photosynthetic protein composition. Changes in the CAO mRNA levels and chlorophyllide a (Chlide a) content in ecotypes and ch1 mutants indicated their significant role in the adjustment mechanism of the photosynthetic apparatus to low-light conditions. Exposure of mutants with a lower chlorophyll b content to short-term (1LL) and long-term low-light stress (10LL) enabled showing a shift in the structure of the PSI and PSII complexes via spectral analysis and the thylakoid composition studies. We demonstrated that both ecotypes, Col-1 and Ler-0, reacted to high-light (HL) conditions in a way remarkably resembling the response of ch1 mutants to normal (NL) conditions. We also presented possible ways of regulating the conversion of chlorophyll a to b depending on the type of light stress conditions.

In Vitro Hydrolysis of Zinc Chlorophyllide a Homologues by a BciC Enzyme.

Chlorosomes in green photosynthetic bacteria are the largest and most efficient light-harvesting antenna systems of all phototrophs. The core part of chlorosomes consists of bacteriochlorophyll c, d, or e molecules. In their biosynthetic pathway, a BciC enzyme catalyzes the removal of the C132-methoxycarbonyl group of chlorophyllide a. In this study, the in vitro enzymatic reactions of chlorophyllide a analogues, C132-methylene- and ethylene-inserted zinc complexes, were examined using a BciC protein from Chlorobaculum tepidum. As the products, their hydrolyzed free carboxylic acids were observed without the corresponding demethoxycarbonylated compounds. The results showed that the in vivo demethoxycarbonylation of chlorophyllide a by an action of the BciC enzyme would occur via two steps: (1) an enzymatic hydrolysis of a methyl ester at the C132-position, followed by (2) a spontaneous (nonenzymatic) decarboxylation in the resulting carboxylic acid.

Chlorophyllide a oxidoreductase Preferentially Catalyzes 8-Vinyl Reduction over B-Ring Reduction of 8-Vinyl Chlorophyllide a in the Late Steps of Bacteriochlorophyll Biosynthesis.

Bacteriochlorophyll a (BChl) is an essential pigment for anoxygenic photosynthesis. In late steps of the BChl biosynthesis of Rhodobacter capsulatus, the C8 vinyl group and C7=C8 double bond of 8-vinyl chlorophyllide a (8 V-Chlide) are reduced by a C8 vinyl reductase (8VR), BciA, and a nitrogenase-like enzyme, chlorophyllide a oxidoreductase (COR), respectively, to produce 3-vinyl-bacteriochlorphyllide a. Recently, we discovered 8VR activity in COR. However, the kinetic parameters of the COR 8VR activity remain unknown, while those of the COR C7=C8 reductase activity and BciA have been reported. Here, we determined the kinetic parameters of COR 8VR activity by using 8 V-Chlide. The Km value for 8 V-Chlide was 1.4 µM, which is much lower than the 6.2 µM determined for the C7=C8 reduction of Chlide. The kinetic parameters of the dual activities of COR suggest that COR catalyzes the reduction of the C8 vinyl group of 8 V-Chlide preferentially over C7=C8 reduction when both substrates are supplied during BChl biosynthesis.

The impact of chlorophyllin on deoxynivalenol transport across jejunum mucosa explants obtained from adult pigs.

Regardless of the efforts put into preventing or reducing fungal growth, extensive mycotoxin contamination has been reported in animal feeds. In the case of pigs, one of the mycotoxins of major concern is deoxynivalenol (DON). The use of adsorbents as feed additives represents one of the strategies to control mycotoxins' contamination in feedstuff. Therefore, the aim of the study was to verify the ability of chlorophyllin (CHL) to reduce the absorption rate of DON in swine mucosa explants. Intestine was obtained from routinely slaughtered adult pigs. The mucosa explants were studied by means of Ussing chamber technique. The effect of DON (10 and 30 µg/ml) on mucosa viability and permeability and CHL (100 µg/ml) impact on DON (30 µg/ml) absorption was verified. The results revealed that mucosa explants isolated from adult animals remained unaffected for 90 min in the presence of DON in the lower concentration (10 µg/ml). Mycotoxin in the higher dose (30 µg/ml) increased mucosa permeability (decreased transepithelial electrical resistance value) and enhanced paracellular transport of lucifer yellow and mannitol but did not affect lactate dehydrogenase leakage. The introduction of CHL neither diminished the absorption rate of DON across swine mucosa explants nor prevented the toxic effects of DON on intestine. In conclusion, the results confirm the negative effect of DON on pig jejunum mucosa. However, the toxic effect of DON was observed only when it was used in relatively high doses. A promising adsorbent agent, CHL, failed to reduce the intensity of DON transport across intestine under in vitro conditions.

Distinct UV-A or UV-B irradiation induces protochlorophyllide photoreduction and bleaching in dark-grown pea (Pisum sativum L.) epicotyls.

The effects of distinct UV-A and UV-B radiations were studied on etiolated pea (Pisum sativum L.) epicotyls. Emission spectra of the native protochlorophyll and protochlorophyllide forms were measured when epicotyls were excited with 360 or 300 nm light. The UV-A (360 nm) excited mainly the non-enzyme-bound monomers of protochlorophyll and protochlorophyllide and the UV-B (300 nm) excited preferentially the flash-photoactive protochlorophyllide complexes. These latter complexes converted into short- and long-wavelength chlorophyllide forms at 10-s illumination with both wavelength irradiations. As the spectral changes were very small, the effects of longer illumination periods were studied. Room temperature fluorescence emission spectra were measured from the same epicotyl spots before and after irradiation with various wavelengths between 280 and 360 nm for 15 min and the "illuminated" minus "dark" difference spectra were calculated. Both the UV-A and the UV-B irradiations caused photoreduction of protochlorophyllide into chlorophyllide. At 10 µmol photons m-2 s-1, the photoreduction rates were similar, however, at 60 µmol photons m-2 s-1, the UV-B irradiation was more effective in inducing chlorophyllide formation than the UV-A. The action spectra of protochlorophyllide plus protochlorophyll loss and chlorophyllide production showed that the radiation around 290 nm was the most effective in provoking protochlorophyllide photoreduction and the UV light above 320 nm caused strong bleaching. These results show that the effect of the UV radiation should be considered when discussing the protochlorophyllide-chlorophyllide photoreduction during germination and as a part of the regeneration of the photosynthetic apparatus proceeding in the daily run of photosynthesis.

Digalactosyldiacylglycerol Is Essential for Organization of the Membrane Structure in Etioplasts.

Angiosperms germinated in the dark develop etioplasts, the chloroplast precursors, in cotyledon cells. Etioplasts contain lattice membrane structures called prolamellar bodies (PLBs) and lamellar prothylakoids as internal membrane systems. PLBs accumulate the chlorophyll intermediate protochlorophyllide (Pchlide) in a complex with NADPH and light-dependent NADPH:Pchlide oxidoreductase (LPOR). Two galactolipids, monogalactosyldiacylglycerol and digalactosyldiacylglycerol (DGDG), are major constituents of etioplast membranes. We previously reported that monogalactosyldiacylglycerol facilitates the synthesis of Pchlide and the formation of the Pchlide-LPOR-NADPH complex in etioplasts, but the importance of DGDG in etioplasts is still unknown. To determine the role of DGDG in etioplast development and functions, we characterized a knockout mutant (dgd1) of Arabidopsis (Arabidopsis thaliana) DGD1, which encodes the major isoform of DGDG synthase, in the etioplast development stage. In etiolated dgd1 seedlings, DGDG content decreased to 20% of the wild-type level, the lattice structure of PLBs was disordered, and the development of prothylakoids was impaired. In addition, membrane-associated processes of Pchlide biosynthesis, formation of the Pchlide-LPOR-NADPH complex, and dissociation of the complex after the photoconversion of Pchlide to chlorophyllide were impaired in dgd1, although the photoconversion reaction by LPOR was not affected by the DGDG deficiency. Total carotenoid content also decreased in etiolated dgd1 seedlings, but the carotenoid composition was unchanged. Our data demonstrate a deep involvement of DGDG in the formation of the internal membrane structures in etioplasts as well as in membrane-associated processes of pigment biosynthesis and pigment-protein complex organization.

Identification of a Chlorophyll Dephytylase Involved in Chlorophyll Turnover in Arabidopsis.

Chlorophyll turns over in green organs during photosystem repair and is salvaged via de- and rephytylation, but the enzyme involved in dephytylation is unknown. We have identified an Arabidopsis thaliana thylakoid protein with a putative hydrolase domain that can dephytylate chlorophyll in vitro and in vivo. The corresponding locus, CHLOROPHYLL DEPHYTYLASE1 (CLD1), was identified by mapping a semidominant, heat-sensitive, missense allele (cld1-1). CLD1 is conserved in oxygenic photosynthetic organisms, sharing structural similarity with pheophytinase, which functions in chlorophyll breakdown during leaf senescence. Unlike pheophytinase, CLD1 is predominantly expressed in green organs and can dephytylate chlorophyll in vitro. The specific activity is significantly higher for the mutant protein encoded by cld1-1 than the wild-type enzyme, consistent with the semidominant nature of the cld1-1 mutation. Supraoptimal CLD1 activities in cld1-1 mutants and transgenic seedlings led to the proportional accumulation of chlorophyllides derived from chlorophyll dephytylation after heat shock, which resulted in light-dependent cotyledon bleaching. Reducing CLD1 expression diminished thermotolerance and the photochemical efficiency of photosystem II under prolonged moderate heat stress. Taken together, our results suggest that CLD1 is the long-sought enzyme for removing the phytol chain from chlorophyll during its turnover at steady state within the chloroplast.

Nano-encapsulated chlorophyllin significantly delays progression of lung cancer both in in vitro and in vivo models through activation of mitochondrial signaling cascades and drug-DNA interaction.

Chlorophyllin (CHL), a sodium-copper-salt derived from chlorophyll, has been widely used as a food-dye, also reportedly having some anti-cancer effect. We tested if PLGA-loaded CHL (NCHL) could have additional protective abilities through its faster and targeted drug delivery in cancer cells. Physico-chemical characterization of NCHL was done through atomic-force microscopy and UV-spectroscopy. NCHL demonstrated greater ability of drug uptake and strong anti-cancer potentials in non-small cell lung cancer cells, A549, as revealed from data of% cell viability, generation of reactive-oxygen-species and expression of bax, bcl2, caspase3, p53 and cytochrome c proteins. Circular dichroic spectral data indicated strong binding of NCHL with calf-thymus-DNA, causing a conformational/structural change in DNA. Further, NCHL could cross the blood-brain-barrier in mice and showed greater efficacy in recovery process of tissue damage, reduction in chromosomal aberrations and% of micronuclei in co-mutagens (Sodiumarsenite+Benzo[a]Pyrene)-treated mice at a much reduced dose, indicating its use in therapeutic oncology.

Lack of Phosphatidylglycerol Inhibits Chlorophyll Biosynthesis at Multiple Sites and Limits Chlorophyllide Reutilization in Synechocystis sp. Strain PCC 6803.

The negatively charged lipid phosphatidylglycerol (PG) constitutes up to 10% of total lipids in photosynthetic membranes, and its deprivation in cyanobacteria is accompanied by chlorophyll (Chl) depletion. Indeed, radioactive labeling of the PG-depleted ΔpgsA mutant of Synechocystis sp. strain PCC 6803, which is not able to synthesize PG, proved the inhibition of Chl biosynthesis caused by restriction on the formation of 5-aminolevulinic acid and protochlorophyllide. Although the mutant accumulated chlorophyllide, the last Chl precursor, we showed that it originated from dephytylation of existing Chl and not from the block in the Chl biosynthesis. The lack of de novo-produced Chl under PG depletion was accompanied by a significantly weakened biosynthesis of both monomeric and trimeric photosystem I (PSI) complexes, although the decrease in cellular content was manifested only for the trimeric form. However, our analysis of ΔpgsA mutant, which lacked trimeric PSI because of the absence of the PsaL subunit, suggested that the virtual stability of monomeric PSI is a result of disintegration of PSI trimers. Interestingly, the loss of trimeric PSI was accompanied by accumulation of monomeric PSI associated with the newly synthesized CP43 subunit of photosystem II. We conclude that the absence of PG results in the inhibition of Chl biosynthetic pathway, which impairs synthesis of PSI, despite the accumulation of chlorophyllide released from the degraded Chl proteins. Based on the knowledge about the role of PG in prokaryotes, we hypothesize that the synthesis of Chl and PSI complexes are colocated in a membrane microdomain requiring PG for integrity.

Broadened Substrate Specificity of 3-Hydroxyethyl Bacteriochlorophyllide a Dehydrogenase (BchC) Indicates a New Route for the Biosynthesis of Bacteriochlorophyll a.

Bacteriochlorophyll a biosynthesis requires formation of a 3-hydroxyethyl group on pyrrole ring A that gets subsequently converted into a 3-acetyl group by 3-vinyl bacteriochlorophyllide a hydratase (BchF) followed by 3-hydroxyethyl bacteriochlorophyllide a dehydrogenase (BchC). Heterologous overproduction of Chlorobaculum tepidum BchF revealed an integral transmembrane protein that was efficiently isolated by detergent solubilization. Recombinant C. tepidum BchC was purified as a soluble protein-NAD(+) complex. Substrate recognition of BchC was investigated using six artificial substrate molecules. Modification of the isocyclic E ring, omission of the central magnesium ion, zinc as an alternative metal ion, and a non-reduced B ring system were tolerated by BchC. According to this broadened in vitro activity, the chlorin 3-hydroxyethyl chlorophyllide a was newly identified as a natural substrate of BchC in a reconstituted pathway consisting of dark-operative protochlorophyllide oxidoreductase, BchF, and BchC. The established reaction sequence would allow for an additional new branching point for the synthesis of bacteriochlorophyll a. Biochemical and site-directed mutagenesis analyses revealed, in contrast to theoretical predictions, a zinc-independent BchC catalysis that requires NAD(+) as a cofactor. Based on these results, we are designating a new medium-chain dehydrogenase/reductase family (MDR057 BchC) as theoretically proposed from a recent bioinformatics analysis.