scholarly journals Protochlorophyllide synthesis by recombinant cyclases from eukaryotic oxygenic phototrophs and the dependence on Ycf54

2020 ◽  
Vol 477 (12) ◽  
pp. 2313-2325 ◽  
Author(s):  
Guangyu E. Chen ◽  
C. Neil Hunter
Keyword(s):  
Protoporphyrin Ix ◽  
Photosynthetic Bacterium ◽  
Normal Function ◽  
Green Algal ◽  
Auxiliary Subunit ◽  
Absolute Requirement ◽  
Model Plant Arabidopsis Thaliana ◽  
Rubrivivax Gelatinosus ◽  
Oxygenic Phototrophs

The unique isocyclic E ring of chlorophylls contributes to their role as light-absorbing pigments in photosynthesis. The formation of the E ring is catalyzed by the Mg-protoporphyrin IX monomethyl ester cyclase, and the O2-dependent cyclase in prokaryotes consists of a diiron protein AcsF, augmented in cyanobacteria by an auxiliary subunit Ycf54. Here, we establish the composition of plant and algal cyclases, by demonstrating the in vivo heterologous activity of O2-dependent cyclases from the green alga Chlamydomonas reinhardtii and the model plant Arabidopsis thaliana in the anoxygenic photosynthetic bacterium Rubrivivax gelatinosus and in the non-photosynthetic bacterium Escherichia coli. In each case, an AcsF homolog is the core catalytic subunit, but there is an absolute requirement for an algal/plant counterpart of Ycf54, so the necessity for an auxiliary subunit is ubiquitous among oxygenic phototrophs. A C-terminal ∼40 aa extension, which is present specifically in green algal and plant Ycf54 proteins, may play an important role in the normal function of the protein as a cyclase subunit.

scholarly journals Magnesium chelatase from Rhodobacter sphaeroides: initial characterization of the enzyme using purified subunits and evidence for a BchI–BchD complex

1999 ◽  
Vol 337 (2) ◽  
pp. 243-251 ◽  
Author(s):  
Lucien C. D. GIBSON ◽  
Poul Erik JENSEN ◽  
C. Neil HUNTER
Keyword(s):  
Rhodobacter Sphaeroides ◽  
Chlorophyll Biosynthesis ◽  
Gel Filtration ◽  
Protoporphyrin Ix ◽  
Photosynthetic Bacterium ◽  
Affinity Tags ◽  
Magnesium Chelatase ◽  
Highly Active ◽  
Mg Chelatase

The enzyme magnesium-protoporphyrin IX chelatase (Mg chelatase) catalyses the insertion of Mg into protoporphyrin IX, the first committed step in (bacterio)chlorophyll biosynthesis. In the photosynthetic bacterium Rhodobacter sphaeroides, this reaction is catalysed by the products of the bchI, bchDand bchH genes. These genes have been expressed in Escherichia coli so that the BchI, BchD and BchH proteins are produced with N-terminal His6 affinity tags, which has led to the production of large amounts of highly purified, highly active Mg chelatase subunits from a single chromatography step. Furthermore, BchD has been purifed free of contamination with the chaperone GroEL, which had proven to be a problem in the past. BchD, present largely as an insoluble protein in E. coli, was purified in 6 M urea and refolded by addition of BchI, MgCl2 and ATP, yielding highly active protein. BchI/BchD mixtures prepared in this way were used in conjunction with BchH to determine the kinetic parameters of R. sphaeroides Mg chelatase for its natural substrates. We have been able to demonstrate for the first time that BchI and BchD form a complex, and that Mg2+ and ATP are required to establish and maintain this complex. Gel filtration data suggest that BchI and BchD form a complex of molecular mass 200 kDa in the presence of Mg2+ and ATP. Our data suggest that, in vivo, BchD is only folded correctly and maintained in its correct conformation in the presence of BchI, Mg2+ and ATP.

scholarly journals Inactivation of Mg Chelatase during Transition from Anaerobic to Aerobic Growth in Rhodobacter capsulatus

2003 ◽  
Vol 185 (11) ◽  
pp. 3249-3258 ◽  
Author(s):  
Robert D. Willows ◽  
Vanessa Lake ◽  
Thomas Hugh Roberts ◽  
Samuel I. Beale
Keyword(s):  
Rhodobacter Capsulatus ◽  
Control Point ◽  
Protoporphyrin Ix ◽  
Photosynthetic Bacterium ◽  
Aerobic Conditions ◽  
Heme Synthesis ◽  
Cell Extracts ◽  
Heterotrophic Metabolism ◽  
Mg Chelatase

ABSTRACT The facultative photosynthetic bacterium Rhodobacter capsulatus can adapt from an anaerobic photosynthetic mode of growth to aerobic heterotrophic metabolism. As this adaptation occurs, the cells must rapidly halt bacteriochlorophyll synthesis to prevent phototoxic tetrapyrroles from accumulating, while still allowing heme synthesis to continue. A likely control point is Mg chelatase, the enzyme that diverts protoporphyrin IX from heme biosynthesis toward the bacteriochlorophyll biosynthetic pathway by inserting Mg2+ to form Mg-protoporphyrin IX. Mg chelatase is composed of three subunits that are encoded by the bchI, bchD, and bchH genes in R. capsulatus. We report that BchH is the rate-limiting component of Mg chelatase activity in cell extracts. BchH binds protoporphyrin IX, and BchH that has been expressed and purified from Escherichia coli is red in color due to the bound protoporphyrin IX. Recombinant BchH is rapidly inactivated by light in the presence of O2, and the inactivation results in the formation of a covalent adduct between the protein and the bound protoporphyrin IX. When photosynthetically growing R. capsulatus cells are transferred to aerobic conditions, Mg chelatase is rapidly inactivated, and BchH is the component that is most rapidly inactivated in vivo when cells are exposed to aerobic conditions. The light- and O2-stimulated inactivation of BchH could account for the rapid inactivation of Mg chelatase in vivo and provide a mechanism for inhibiting the synthesis of bacteriochlorophyll during adaptation of photosynthetically grown cells to aerobic conditions while still allowing heme synthesis to occur for aerobic respiration.

iTSP-PseAAC: Identify Tumor Suppressor Proteins by Using Fully Connected Neural Network and PseAAC

2021 ◽  
Vol 15 ◽  
Author(s):  
Muhammad Awais ◽  
Waqar Hussain ◽  
Nouman Rasool ◽  
Yaser Daanial Khan
Keyword(s):  
Tumor Suppressor ◽  
Cross Validation ◽  
Control Cell ◽  
Normal Function ◽  
In Vivo Studies ◽  
Tumor Suppressor Proteins ◽  
Proposed Model ◽  
Self Consistency

Background: The uncontrolled growth due to accumulation of genetic and epigenetic changes as a result of loss or reduction in the normal function of Tumor Suppressor Genes (TSGs) and Pro-oncogenes is known as cancer. TSGs control cell division and growth by repairing of DNA mistakes during replication and restrict the unwanted proliferation of a cell or activities, those are the part of tumor production. Objectives: This study aims to propose a novel, accurate, user-friendly model to predict tumor suppressor proteins, which would be freely available to experimental molecular biologists to assist them using in vitro and in vivo studies. Methods: The predictor model has used the input feature vector (IFV) calculated from the physicochemical properties of proteins based on FCNN to compute the accuracy, sensitivity, specificity, and MCC. The proposed model was validated against different exhaustive validation techniques i.e. self-consistency and cross-validation. Results: Using self-consistency, the accuracy is 99%, for cross-validation and independent testing has 99.80% and 100% accuracy respectively. The overall accuracy of the proposed model is 99%, sensitivity value 98% and specificity 99% and F1-score was 0.99. Conclusion: It concludes, the proposed model for prediction of the tumor suppressor proteins can predict the tumor suppressor proteins efficiently, but it still has space for improvements in computational ways as the protein sequences may rapidly increase, day by day.

scholarly journals All-Trans Retinoic Acid Increases DRP1 Levels and Promotes Mitochondrial Fission

Cells ◽  
2021 ◽  
Vol 10 (5) ◽  
pp. 1202
Author(s):  
Bojjibabu Chidipi ◽  
Syed Islamuddin Shah ◽  
Michelle Reiser ◽  
Manasa Kanithi ◽  
Amanda Garces ◽  
...  
Keyword(s):  
Retinoic Acid ◽  
Mitochondrial Fission ◽  
Normal Function ◽  
Mitochondrial Network ◽  
Protein Levels ◽  
Trans Retinoic Acid ◽  
All Trans Retinoic Acid ◽  
Area And Perimeter

In the heart, mitochondrial homeostasis is critical for sustaining normal function and optimal responses to metabolic and environmental stressors. Mitochondrial fusion and fission are thought to be necessary for maintaining a robust population of mitochondria, and disruptions in mitochondrial fission and/or fusion can lead to cellular dysfunction. The dynamin-related protein (DRP1) is an important mediator of mitochondrial fission. In this study, we investigated the direct effects of the micronutrient retinoid all-trans retinoic acid (ATRA) on the mitochondrial structure in vivo and in vitro using Western blot, confocal, and transmission electron microscopy, as well as mitochondrial network quantification using stochastic modeling. Our results showed that ATRA increases DRP1 protein levels, increases the localization of DRP1 to mitochondria in isolated mitochondrial preparations. Our results also suggested that ATRA remodels the mitochondrial ultrastructure where the mitochondrial area and perimeter were decreased and the circularity was increased. Microscopically, mitochondrial network remodeling is driven by an increased rate of fission over fusion events in ATRA, as suggested by our numerical modeling. In conclusion, ATRA results in a pharmacologically mediated increase in the DRP1 protein. It also results in the modulation of cardiac mitochondria by promoting fission events, altering the mitochondrial network, and modifying the ultrastructure of mitochondria in the heart.

scholarly journals Chimeric Mouse With Humanized Liver Is an Appropriate Animal Model to Investigate Mode of Action for Porphyria-Mediated Hepatocytotoxicity

2021 ◽  
pp. 019262332110274
Author(s):  
Ayumi Eguchi ◽  
Satoki Fukunaga ◽  
Keiko Ogata ◽  
Masahiko Kushida ◽  
Hiroyuki Asano ◽  
...  
Keyword(s):  
Experimental Model ◽  
Mode Of Action ◽  
Aminolevulinic Acid ◽  
Protoporphyrin Ix ◽  
Human Hepatocytes ◽  
Chimeric Mouse ◽  
Hepatocellular Injury ◽  
Hepatocyte Necrosis ◽  
Key Events

Porphyrinogenic compounds are known to induce porphyria-mediated hepatocellular injury and subsequent regenerative proliferation in rodents, ultimately leading to hepatocellular tumor induction. However, an appropriate in vivo experimental model to evaluate an effect of porphyrinogenic compounds on human liver has not been fully established. Recently, the chimeric mouse with humanized liver (PXB mice) became widely used as a humanized model in which human hepatocytes are transplanted. In the present study, we examined the utility of PXB mice as an in vivo experimental model to evaluate the key events of the porphyria-mediated cytotoxicity mode of action (MOA) in humans. The treatment of PXB mice with 5-aminolevulinic acid, a representative porphyrinogenic compound, for 28 days caused protoporphyrin IX accumulation, followed by hepatocyte necrosis, increased mitosis, and an increase in replicative DNA synthesis in human hepatocytes, indicative of cellular injury and regenerative proliferation, similar to findings in patients with porphyria or experimental porphyria models and corresponding to the key events of the MOA for porphyria-mediated hepatocellular carcinogenesis. We conclude that the PXB mouse is a useful model to evaluate the key events of the porphyria-mediated cytotoxicity MOA in humans and suggest the utility of PXB mice for clarifying the human relevancy of findings in mice.

scholarly journals Anticoagulant Properties of a Green Algal Rhamnan-type Sulfated Polysaccharide and Its Low-molecular-weight Fragments Prepared by Mild Acid Degradation

Marine Drugs ◽  
2018 ◽  
Vol 16 (11) ◽  
pp. 445 ◽  
Author(s):  
Xue Liu ◽  
Peng Du ◽  
Xiao Liu ◽  
Sujian Cao ◽  
Ling Qin ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Anticoagulant Activity ◽  
Sulfated Polysaccharide ◽  
Low Molecular Weight ◽  
Thrombin Time ◽  
Acid Degradation ◽  
Molecular Weights ◽  
Green Algal

The active sulfated polysaccharide from seaweed possesses important pharmaceutical and biomedical potential. In the study, Monostroma sulfated polysaccharide (MSP) was obtained from Monostroma angicava, and the low-molecular-weight fragments of MSP (MSP-Fs: MSP-F1–MSP-F6) were prepared by controlled acid degradation. The molecular weights of MSP and MSP-F1–MSP-F6 were 335 kDa, 240 kDa, 90 kDa, 40 kDa, 24 kDa, 12 kDa, and 6.8 kDa, respectively. The polysaccharides were sulfated rhamnans that consisted of →3)-α-l-Rhap-(1→ and →2)-α-l-Rhap-(1→ units with partial sulfation at C-2 of →3)-α-l-Rhap-(1→ and C-3 of →2)-α-l-Rhap-(1→. Anticoagulant properties in vitro of MSP and MSP-F1–MSP-F6 were evaluated by studying the activated partial thromboplastin time, thrombin time, and prothrombin time. Anticoagulant activities in vivo of MSP and MSP-F4 were further evaluated; their fibrin(ogen)olytic activities in vivo and thrombolytic properties in vitro were also assessed by D-dimer, fibrin degradation products, plasminogen activator inhibitior-1, and clot lytic rate assays. The results showed that MSP and MSP-F1–MSP-F4 with molecular weights of 24–240 kDa had strong anticoagulant activities. A decrease in the molecular weight of MSP-Fs was accompanied by a decrease in the anticoagulant activity, and higher anticoagulant activity requires a molecular weight of over 12 kDa. MSP and MSP-F4 possessed strong anticoagulant activities in vivo, as well as high fibrin(ogen)olytic and thrombolytic activities. MSP and MSP-F4 have potential as drug or helpful food supplements for human health.

The protective properties of synthetic porphyrin tin complexes in toxic hyperbilirubinemia

2000 ◽  
Vol 04 (03) ◽  
pp. 243-247 ◽  
Author(s):  
T. O. PHILIPPOVA ◽  
B. N. GALKIN ◽  
N. YA. GOLOVENKO ◽  
Z. I. ZHILINA ◽  
S. V. VODZINSKII
Keyword(s):  
Heme Oxygenase ◽  
Serum Bilirubin ◽  
Protoporphyrin Ix ◽  
Serum Bilirubin Level ◽  
Oxygenase Activity ◽  
Tetraphenyl Porphyrin ◽  
Tin Complexes ◽  
Cytochrome P 450

Tin complexes of meso-substituted synthetic porphyrins, namely Sn 4+-meso-tetraphenyl- porphyrin ( Sn - TPP ) and Sn 4+-meso-tetrakis(N-methyl-3-pyridyl)porphyrin tetratosylate ( Sn - TMe -3- PyP ), efficiently decrease the serum bilirubin level when injected subcutaneously at a dose of 100 μM kg−1 body weight into mice. These compounds are active during hyperbilirubinemia, induced by phenylhydrazine, hemin and tetrachloromethane, and also during autoimmune hemolytic anemia. In the latter case a decrease in serum bilirubin content was observed, as well as a decrease in the amount of blood reticulocytes which reflects a milder course of the disease. The Sn complexes under study induce, in vivo, cytochrome P-450, inhibit microsomal heme oxygenase and decrease the intensity of lipid peroxidation. At the same time, in vitro the hepatic and splenic heme oxygenase activity is blocked only when a 0.1 μM concentration of Sn - TMe -3- PyP or Sn -protoporphyrin IX is added to the incubation mixture. Sn - TPP does not affect the activity of this enzyme in vitro.

scholarly journals Phospholipid and cation activation of chimaeric choline/ethanolamine phosphotransferases

1996 ◽  
Vol 313 (3) ◽  
pp. 729-735 ◽  
Author(s):  
Christopher R. McMASTER ◽  
Sherry C. MORASH ◽  
Robert M. BELL
Keyword(s):  
Tertiary Structure ◽  
Feedback Inhibition ◽  
Head Group ◽  
Biologically Relevant ◽  
Activation Domains ◽  
Cation Activation ◽  
Absolute Requirement ◽  
Insight Into

The Saccharomyces cerevisiae CPT1 and EPT1 genes encode for a cholinephosphotransferase (CPT) and choline/ ethanolaminephosphotransferase, respectively. Both Cpt1p and Ept1p activities display an absolute requirement for cations and phospholipids. A mixed-micelle assay was employed to determine cation and lipid activators of parental and chimaeric Cpt1p/ Ept1p enzymes to gain insight into their mechanism(s) of activation. Mg2+, Mn2+ and Co2+ were the only cations capable of activating Cpt1p and Ept1p in vitro. Kinetic data revealed that only Mg2+ is present in appropriate amounts to activate CPT activity in vivo. The two enzymes displayed distinct activation profiles on the basis of their relative affinities for Mg2+, Mn2+ and Co2+. This allowed the use of chimaeric enzymes to determine the mechanism of cation activation. Cations do not activate Cpt1p or Ept1p by complexing with the substrate, CDP-choline, but instead bind to disparate regions within the enzymes themselves. Cpt1p and Ept1p also displayed distinct phospholipid activation profiles. Phospholipid activation required a phosphate and/or glycero-phosphoester linkage, with the phospho-head group moiety positioned at the surface of the micelle. Assays with parental and chimaeric Cpt1p/Ept1p constructs revealed that the phospholipid binding/activation domains are not located within linear segments of the protein, but instead are contained within distinct and separate regions of the proteins that require an intact tertiary structure for formation. Phosphatidylcholine (and its structural analogue sphingomyelin) were the best lipid activators of Cpt1p, the main biologically relevant CPT activity in S. cerevisiae. Hence CPT displays product activation. Because phosphatidylcholine is an efficient activator of CPT activity (and hence Cpt1p is not subject to feedback inhibition by its product), Cpt1p is incapable of functioning as a direct monitor of membrane phosphatidylcholine composition.

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