The synthesis of magnesium-protoporphyrin IX by etiochloroplast membrane preparations

Richter, Mark L.; Rienits, K.G.

doi:10.1016/0304-4165(82)90177-5

Cited by 26 publications

(17 citation statements)

References 13 publications

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“…2) is paralleled by a decrease in Mg-chelatase activity, indicating that the two factors are closely related. We have assayed Mg-chelatase activity in osmotically lysed and freeze/thawed plastids and have observed very low activities, comparable with those reported by Richter and Reinits (25). Attempts to stabilize this activity in our preparations have been unsuccessful.…”

Section: Regulation Of Mg-chelatase Activity By Greening and Putativesupporting

confidence: 79%

“…In an attempt to simplify the Mg-chelatase assay system, Richter and Rienits (25) ruptured cucumber plastids by osmotic lysis and removed the soluble intraplastid contents by buffer washes. They were able to measure low levels of Mgchelatase activity by using a ['4C]Proto substrate; however, this specific activity was only 2.5% of the activity of intact chloroplasts.…”

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confidence: 99%

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Further Characterization of the Magnesium Chelatase in Isolated Developing Cucumber Chloroplasts

Walker¹,

Weinstein²

1991

Plant Physiol.

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Mg-chelatase catalyzes the first step unique to the chlorophyll branch of tetrapyrrole biosynthesis, namely the insertion of Mg into protoporphyrin IX (Proto). Mg-chelatase was assayed in intact chloroplasts from semi-green cucumber (Cucumis sativus, cv Sumter) cotyledons. In the presence of Proto and MgATP, enzyme activity was linear for 50 minutes. Plastid intactness was directly related to (and necessary for) Mg-chelatase activity. Uncouplers and ionophores did not inhibit Mg-chelatase in the presence of ATP. The nonhydrolyzable ATP analogs, O,-y-methylene ATP and adenylylimidodiphosphate, could not sustain Mg-chelatase activity alone and were inhibitory in the presence of ATP (lso 10 and 3 millimolar, respectively). Mg-chelatase was also inhibited by N-ethylmaleimide (l,o, 50 micromolar) and the metal ion chelators

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Section: Regulation Of Mg-chelatase Activity By Greening and Putativesupporting

confidence: 79%

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confidence: 99%

Further Characterization of the Magnesium Chelatase in Isolated Developing Cucumber Chloroplasts

Walker¹,

Weinstein²

1991

Plant Physiol.

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“…First, activity could be measured in chloroplasts (higher plants) or whole cells (bacteria), but breakage of the chloroplast or cell invariably resulted in either a complete loss or approx. 98 % loss of activity [12][13][14][15][16][17]. Secondly, in these systems, Mgchelation was only observed in the presence of ATP [13,15].…”

Section: Demonstration Of Mg-chelatase Activity In Vitromentioning

confidence: 99%

Mechanism and regulation of Mg-chelatase

Walker

Willows

1997

Biochemical Journal

229

173

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Mg-chelatase catalyses the insertion of Mg into protoporphyrin IX (Proto). This seemingly simple reaction also is potentially one of the most interesting and crucial steps in the (bacterio)chlorophyll (Bchl/Chl)-synthesis pathway, owing to its position at the branch-point between haem and Bchl/Chl synthesis. Up until the level of Proto, haem and Bchl/Chl synthesis share a common pathway. However, at the point of metal-ion insertion there are two choices: Mg2+ insertion to make Bchl/Chl (catalysed by Mg-chelatase) or Fe2+ insertion to make haem (catalysed by ferrochelatase). Thus the relative activities of Mg-chelatase and ferrochelatase must be regulated with respect to the organism's requirements for these end products . How is this regulation achieved? For Mg-chelatase, the recent design of an in vitro assay combined with the identification of Bchl-biosynthetic enzyme genes has now made it possible to address this question. In all photosynthetic organisms studied to date, Mg-chelatase is a three-component enzyme, and in several species these proteins have been cloned and expressed in an active form. The reaction takes place in two steps, with an ATP-dependent activation followed by an ATP-dependent chelation step. The activation step may be the key to regulation, although variations in subunit levels during diurnal growth may also play a role in determining the flux through the Bchl/Chl and haem branches of the pathway.

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“…The chelation of magnesium into protoporphyrin TX has been demonstrated using intact cells of R. sphaeroides (Gorchein, 1972,1973), isolated plastids (Castelfranco et al, 1979;Pardo et al, 1980;Fuesler et al, 1981 ;Richter and Rienits, 1980) and broken plastid systems (Richter and Rienits, 1982;Weinstein, 1991, 1994;Walker et al, 1992;Lee et al, 1992). In these systems, ATP is absolutely required and it has been demonstrated for pea (Pisum sativum) chloroplasts that two protein fractions participate in the enzymic reaction and that ATP is required for both activation of the proteins and Mg2+ chelation .…”

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Three Separate Proteins Constitute the Magnesium Chelatase of Rhodobacter Sphaeroides

Willows

Gibson

Kanangara

et al. 1996

European Journal of Biochemistry

118

139

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The insertion of magnesium into protoporphyrin IX is the first step unique to chlorophyll production and is catalyzed by magnesium chelatase. The Rhodobacter sphaeroides genes, bchl and bchD together, and bchH alone, were cloned and expressed with the pET3a vector in Escherichia coli strain BL21 (DE3). The 40-kDa BchI protein was synthesized in greater abundance compared to the 70-kDa BchD protein when both were expressed together from the same plasmid. The production of large amounts of the 140-kDa BchH protein in E. coli was accompanied by an accumulation of protoporphyrin TX. The accumulated protoporphyrin IX was bound specifically to BchH in an approximate molar ratio of 1 : l . All three recombinant proteins were soluble; BchH was monomeric, BchI was dimeric, while BchD appeared to be polymeric with a molecular mass of approximately 550 kDa. The BchH and BchT proteins were purified to apparent homogeneity while BchD was separated from BchI and partially purified. Magnesium was inserted into protoporphyrin IX and deuteroporphyrin by combining these three proteins in the presence of ATP. One monomer of BchH to one dimer of BchI gave the optimal magnesium chelatase activity and the activity was dependent on the amount of partially purified BchD added to the assay at the optimum BchH:BchI ratio. The reaction was dissected into two parts with an activation step requiring BchI, BchD, and Mg2 ' -ATP, and a metal-insertion step which in addition requires MgZ ' , protoporphyrin IX, and BchH.The stoichiometric binding of protoporphyrin IX to BchH in vitro is direct evidence for BchH carrying out such a role in vivo whereas the other two proteins are involved in ATP activation and magnesium insertion.

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The synthesis of magnesium-protoporphyrin IX by etiochloroplast membrane preparations

Cited by 26 publications

References 13 publications

Further Characterization of the Magnesium Chelatase in Isolated Developing Cucumber Chloroplasts

Further Characterization of the Magnesium Chelatase in Isolated Developing Cucumber Chloroplasts

Mechanism and regulation of Mg-chelatase

Three Separate Proteins Constitute the Magnesium Chelatase of Rhodobacter Sphaeroides

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