Mg-chelatase catalyzes the insertion of Mg 2þ into protoporphyrin IX at the first committed step of the chlorophyll biosynthetic pathway. It consists of three subunits: I, D, and H. The I subunit belongs to the AAA protein superfamily (ATPases associated with various cellular activities) that is known to form hexameric ring structures in an ATP-dependant fashion. Dominant mutations in the I subunit revealed that it functions in a cooperative manner. We demonstrated that the D subunit forms ATP-independent oligomeric structures and should also be classified as an AAA protein. Furthermore, we addressed the question of cooperativity of the D subunit with barley (Hordeum vulgare) mutant analyses. The recessive behavior in vivo was explained by the absence of mutant proteins in the barley cell. Analogous mutations in Rhodobacter capsulatus and the resulting D proteins were studied in vitro. Mixtures of wild-type and mutant R. capsulatus D subunits showed a lower activity compared with wild-type subunits alone. Thus, the mutant D subunits displayed dominant behavior in vitro, revealing cooperativity between the D subunits in the oligomeric state. We propose a model where the D oligomer forms a platform for the stepwise assembly of the I subunits. The cooperative behavior suggests that the D oligomer takes an active part in the conformational dynamics between the subunits of the enzyme.
a b s t r a c tOryza sativa GUN4 together with the magnesium chelatase subunits ChlI, ChlD, and ChlH have been heterologously expressed and purified to reconstitute magnesium chelatase activity in vitro. Maximum magnesium chelatase activity requires pre-activation of OsChlH with OsGUN4, Mg 2+ and protoporphyrin-IX. OsGUN4 and OsChlH preincubated without protoporphyrin-IX yields magnesium chelatase activity similar to assays without OsGUN4, suggesting formation of a dead-end complex. Either 9 or 10 C-terminal amino acids of OsGUN4 are slowly hydrolyzed to yield a truncated OsGUN4. These truncated OsGUN4 still bind protoporphyrin-IX and Mg-protoporphyrin-IX but are unable to activate OsChlH. This suggests the mechanism of GUN4 activation of magnesium chelatase is different in eukaryotes compared to cyanobacteria as the orthologous cyanobacterial GUN4 proteins lack this C-terminal extension.
The genomes uncoupled 4 (GUN4) protein is a nuclear-encoded, chloroplast-localized, porphyrin-binding protein implicated in retrograde signaling between the chloroplast and nucleus, although its exact role in this process is still unclear. Functionally, it enhances Mg-chelatase activity in the chlorophyll biosynthesis pathway. Because GUN4 is present only in organisms that carry out oxygenic photosynthesis and because it binds protoporphyrin IX (PPIX) and Mg-PPIX, it has been suggested that it prevents production of light-and PPIX-or Mg-PPIX-dependent reactive oxygen species. A chld-1/GUN4 mutant with elevated PPIX has a light-dependent up-regulation of GUN4, implicating this protein in light-dependent sensing of PPIX, with the suggestion that GUN4 reduces PPIX-generated singlet oxygen, O 2 (a 1 ⌬ g ), and subsequent oxidative damage
The metabolic pathway known as (bacterio)chlorophyll biosynthesis is initiated by magnesium chelatase (BchI, BchD, BchH). This first step involves insertion of magnesium into protoporphyrin IX (proto), a process requiring ATP hydrolysis. Structural information shows that the BchI and BchD subunits form a double hexameric enzyme complex, whereas BchH binds proto and can be purified as BchH-proto. We utilized the Rhodobacter capsulatus magnesium chelatase subunits using continuous magnesium chelatase assays and treated the BchD subunit as the enzyme with both BchI and BchH-proto as substrates. Michaelis-Menten kinetics was observed with the BchI subunit, whereas the BchH subunit exhibited sigmoidal kinetics (Hill coefficient of 1.85). The BchI⅐BchD complex had intrinsic ATPase activity, and addition of BchH greatly increased ATPase activity. This was concentration-dependent and gave sigmoidal kinetics, indicating there is more than one binding site for the BchH subunit on the BchI⅐BchD complex. ATPase activity was ϳ40-fold higher than magnesium chelatase activity and continued despite cessation of magnesium chelation, implying one or more secondary roles for ATP hydrolysis and possibly an as yet unknown switch required to terminate ATPase activity. One of the secondary roles for BchH-stimulated ATP hydrolysis by a BchI⅐BchD complex is priming of BchH to facilitate correct binding of proto to BchH in a form capable of participating in magnesium chelation. This porphyrin binding is the rate-limiting step in catalysis. These data suggest that ATP hydrolysis by the BchI⅐BchD complex causes a series of conformational changes in BchH to effect substrate binding, magnesium chelation, and product release.
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 His(6)-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).
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