scholarly journals S-Adenosyl-L-methionine:magnesium-protoporphyrin IX O-methyltransferase from Rhodobacter capsulatus: mechanistic insights and stimulation with phospholipids

2007 ◽  
Vol 406 (3) ◽  
pp. 469-478 ◽  
Author(s):  
Artur Sawicki ◽  
Robert D. Willows
Keyword(s):  
Enzymatic Activity ◽  
Inclusion Bodies ◽  
Rhodobacter Capsulatus ◽  
Protoporphyrin Ix ◽  
Size Exclusion ◽  
Zinc Protoporphyrin ◽  
Sequential Reaction ◽  
Magnesium Chelatase ◽  
Heat Stable ◽  
Exclusion Chromatography

The enzyme BchM (S-adenosyl-L-methionine:magnesium-protoporphyrin IX O-methyltransferase) from Rhodobacter capsulatus catalyses an intermediate reaction in the bacteriochlorophyll biosynthetic pathway. Overexpression of His6-tagged protein in Escherichia coli resulted in the majority of polypeptide existing as inclusion bodies. Purification from inclusion bodies was performed using metal-affinity chromatography after an elaborate wash step involving surfactant polysorbate-20. Initial enzymatic assays involved an in situ generation of S-adenosyl-L-methionine substrate using a crude preparation of S-adenosyl-L-methionine synthetase and this resulted in higher enzymatic activity compared with commercial S-adenosyl-L-methionine. A heat-stable stimulatory component present in the S-adenosyl-L-methionine synthetase was found to be a phospholipid, which increased enzymatic activity 3–4-fold. Purified phospholipids also stabilized enzymatic activity and caused a disaggregation of the protein to lower molecular mass forms, which ranged from monomeric to multimeric species as determined by size-exclusion chromatography. There was no stimulatory effect observed with magnesium–chelatase subunits on methyltransferase activity using His–BchM that had been stabilized with phospholipids. Substrate specificity of the enzyme was limited to 5-co-ordinate square-pyramidal metalloporphyrins, with magnesium-protoporphyrin IX being the superior substrate followed by zinc-protoporphyrin IX and magnesium-deuteroporphyrin. Kinetic analysis indicated a random sequential reaction mechanism. Three non-substrate metalloporphyrins acted as inhibitors with different modes of inhibition exhibited with manganese III-protoporphyrin IX (non-competitive or uncompetitive) compared with cobalt II-protoporphyrin IX (competitive).

scholarly journals ATPase activity associated with the magnesium chelatase H-subunit of the chlorophyll biosynthetic pathway is an artefact

2006 ◽  
Vol 400 (3) ◽  
pp. 477-484 ◽  
Author(s):  
Nick Sirijovski ◽  
Ulf Olsson ◽  
Joakim Lundqvist ◽  
Salam Al-Karadaghi ◽  
Robert D. Willows ◽  
...  
Keyword(s):  
Atpase Activity ◽  
Molecular Mass ◽  
Mass Fraction ◽  
Biosynthetic Pathway ◽  
Rhodobacter Capsulatus ◽  
Gel Filtration ◽  
Protoporphyrin Ix ◽  
High Molecular Mass ◽  
Magnesium Chelatase ◽  
Ring Complex

Magnesium chelatase inserts Mg2+ into protoporphyrin IX and is the first unique enzyme of the chlorophyll biosynthetic pathway. It is a heterotrimeric enzyme, composed of I- (40 kDa), D- (70 kDa) and H- (140 kDa) subunits. The I- and D-proteins belong to the family of AAA+ (ATPases associated with various cellular activities), but only I-subunit hydrolyses ATP to ADP. The D-subunits provide a platform for the assembly of the I-subunits, which results in a two-tiered hexameric ring complex. However, the D-subunits are unstable in the chloroplast unless ATPase active I-subunits are present. The H-subunit binds protoporphyrin and is suggested to be the catalytic subunit. Previous studies have indicated that the H-subunit also has ATPase activity, which is in accordance with an earlier suggested two-stage mechanism of the reaction. In the present study, we demonstrate that gel filtration chromatography of affinity-purified Rhodobacter capsulatus H-subunit produced in Escherichia coli generates a high- and a low-molecular-mass fraction. Both fractions were dominated by the H-subunit, but the ATPase activity was only found in the high-molecular-mass fraction and magnesium chelatase activity was only associated with the low-molecular-mass fraction. We demonstrated that light converted monomeric low-molecular-mass H-subunit into high-molecular-mass aggregates. We conclude that ATP utilization by magnesium chelatase is solely connected to the I-subunit and suggest that a contaminating E. coli protein, which binds to aggregates of the H-subunit, caused the previously reported ATPase activity of the H-subunit.

scholarly journals Bilin-dependent regulation of chlorophyll biosynthesis by GUN4

2021 ◽  
Vol 118 (20) ◽  
pp. e2104443118
Author(s):  
Weiqing Zhang ◽  
Robert D. Willows ◽  
Rui Deng ◽  
Zheng Li ◽  
Mengqi Li ◽  
...  
Keyword(s):  
Enzymatic Activity ◽  
Heme Oxygenase ◽  
Chlorophyll Biosynthesis ◽  
Protoporphyrin Ix ◽  
Common Trunk ◽  
Magnesium Chelatase ◽  
Photosynthetic Eukaryotes ◽  
Linear Tetrapyrroles ◽  
Oxygenic Phototrophs ◽  
Binding Component

Biosyntheses of chlorophyll and heme in oxygenic phototrophs share a common trunk pathway that diverges with insertion of magnesium or iron into the last common intermediate, protoporphyrin IX. Since both tetrapyrroles are pro-oxidants, it is essential that their metabolism is tightly regulated. Here, we establish that heme-derived linear tetrapyrroles (bilins) function to stimulate the enzymatic activity of magnesium chelatase (MgCh) via their interaction with GENOMES UNCOUPLED 4 (GUN4) in the model green alga Chlamydomonas reinhardtii. A key tetrapyrrole-binding component of MgCh found in all oxygenic photosynthetic species, CrGUN4, also stabilizes the bilin-dependent accumulation of protoporphyrin IX-binding CrCHLH1 subunit of MgCh in light-grown C. reinhardtii cells by preventing its photooxidative inactivation. Exogenous application of biliverdin IXα reverses the loss of CrCHLH1 in the bilin-deficient heme oxygenase (hmox1) mutant, but not in the gun4 mutant. We propose that these dual regulatory roles of GUN4:bilin complexes are responsible for the retention of bilin biosynthesis in all photosynthetic eukaryotes, which sustains chlorophyll biosynthesis in an illuminated oxic environment.

1-N-histidine phosphorylation of ChlD by the AAA+ ChlI2 stimulates magnesium chelatase activity in chlorophyll synthesis

2017 ◽  
Vol 474 (12) ◽  
pp. 2095-2105 ◽  
Author(s):  
Artur Sawicki ◽  
Shuaixiang Zhou ◽  
Kathrin Kwiatkowski ◽  
Meizhong Luo ◽  
Robert D. Willows
Keyword(s):  
Rhodobacter Capsulatus ◽  
Protoporphyrin Ix ◽  
Chlorophyll Synthesis ◽  
Magnesium Chelatase ◽  
Orthologous Protein ◽  
Substrate Complex ◽  
Motor Complex ◽  
Histidine Phosphorylation ◽  
Mg Chelatase ◽  
Stimulation Of

Magnesium chelatase (Mg-chelatase) inserts magnesium into protoporphyrin during the biosynthesis of chlorophyll and bacteriochlorophyll. Enzyme activity is reconstituted by forming two separate preactivated complexes consisting of a GUN4/ChlH/protoporphyrin IX substrate complex and a ChlI/ChlD enzyme ‘motor’ complex. Formation of the ChlI/ChlD complex in both Chlamydomonas reinhardtii and Oryza sativa is accompanied by phosphorylation of ChlD by ChlI, but the orthologous protein complex from Rhodobacter capsulatus, BchI/BchD, gives no detectable phosphorylation of BchD. Phosphorylation produces a 1-N-phospho-histidine within ChlD. Proteomic analysis indicates that phosphorylation occurs at a conserved His residue in the C-terminal integrin I domain of ChlD. Comparative analysis of the ChlD phosphorylation with enzyme activities of various ChlI/ChlD complexes correlates the phosphorylation by ChlI2 with stimulation of Mg-chelatase activity. Mutation of the H641 of CrChlD to E641 prevents both phosphorylation and stimulation of Mg-chelatase activity, confirming that phosphorylation at H641 stimulates Mg-chelatase. The properties of ChlI2 compared with ChlI1 of Chlamydomonas and with ChlI of Oryza, shows that ChlI2 has a regulatory role in Chlamydomonas.

scholarly journals PTD-mediated delivery of α-globin chain into Κ-562 erythroleukemia cells and α-thalassemic (HBH) patients’ RBCs ex vivo in the frame of Protein Replacement Therapy

2021 ◽  
Vol 28 (1) ◽  
Author(s):  
Androulla N. Miliotou ◽  
Dionysia Papagiannopoulou ◽  
Efthymia Vlachaki ◽  
Martina Samiotaki ◽  
Dimitra Laspa ◽  
...  
Keyword(s):  
Replacement Therapy ◽  
Inclusion Bodies ◽  
Ex Vivo ◽  
Research Work ◽  
Size Exclusion ◽  
Globin Chain ◽  
Therapy Approach ◽  
Globin Chains ◽  
Exclusion Chromatography

Abstract Background α-Thalassemia, a congenital hemoglobinopathy, is characterized by deficiency and/or reduced levels of α-globin chains in serious forms of α-thalassemia (HbH disease/Hb Bart’s). This research work deals with a Protein Replacement Therapy approach in order to manage α-thalassemia manifestations, caused by the excess of β-globin chain into HbH RBCs. The main goal was to produce the recombinant human α-globin chain in fusion with TAT, a Protein Transduction Domain, to ex vivo deliver it into HbH patients RBCs, to replace the endogenous missing α-globin chain. Results Cloning of the α-globin coding sequence, fused to the nucleotide sequence of TAT peptide was conducted and the human recombinant fusion proteins, 10xHis-XaSITE-α-globin-HA and 10xHis-XaSITE-TAT-α-globin-HA were produced. The ability of human recombinant 10xHis-XaSITE-α-globin-HA to interact in vitro with the previously produced 10xHis-XaSITE-TAT-β-globin-HA and form α-/β-globin heterodimers, was assessed and confirmed by size exclusion chromatography. The recombinant 10xHis-XaSITE-TAT-α-globin-HA was successfully delivered into human proerythroid K-562 cells, during the preliminary transduction evaluation experiments. Finally, the recombinant, TAT-fused α-globin was successfully transduced into RBCs, derived from HbH patients and reduced the formation of HbH-Inclusion Bodies, known to contain harmful β4-globin chain tetramers. Conclusions Our data confirm the successful ex vivo transduction of recombinant α-globin chains in HbH RBCs to replace the missing a-globin chain and reduce the HbH-inclusion bodies, seen in α-thalassemias. These findings broaden the possibility of applying a Protein Replacement Therapy approach to module sever forms of α-thalassemia, using recombinant α-globin chains, through PTD technology.

scholarly journals Separation of native and truncated forms of poliovirus protease 3C produced in Escherichia coli

1993 ◽  
Vol 290 (3) ◽  
pp. 797-800 ◽  
Author(s):  
L Polgár ◽  
F Erdélyi ◽  
E Hajnal ◽  
M Löw ◽  
L Gráf ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Inclusion Bodies ◽  
Initiation Site ◽  
Full Length ◽  
Size Exclusion ◽  
Truncated Form ◽  
Buffer Solution ◽  
Diluted Solution ◽  
Exclusion Chromatography ◽  
Internal Initiation

Poliovirus protease 3C is a cysteine enzyme that is essential for the processing of the viral precursor polyprotein containing structural proteins and enzymes, including the protease itself. We have constructed the plasmid pSD/PV3C which produced protease 3C as inclusion bodies when expressed in Escherichia coli. In addition to the full-length protease, a truncated form was also generated, starting from an internal initiation site (Met-27). The enzyme was renatured by dilution of a 6 M guanidinium chloride solution of the inclusion bodies, and the proteins were precipitated from the diluted solution with ammonium sulphate. By extracting the precipitate with a buffer solution, the full-length enzyme could be completely separated from its N-terminally truncated form. Size-exclusion chromatography of the extracted protease 3C resulted in an active enzyme which appeared homogeneous by SDS/PAGE. For measuring the activity of the protease, a spectrofluorimetric method was devised to monitor the hydrolysis continuously, which is simpler and more precise than the h.p.l.c. technique used previously.

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