The synthesis of magnesium and zinc protoporphyrin IX and their monomethyl esters in etioplast preparations studied by high pressure liquid chromatography

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

doi:10.1016/0014-5793(80)80646-6

Cited by 16 publications

(5 citation statements)

References 15 publications

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“…The physiological substrate Proto was also active in the reconstituted system (Table 3). There have been reports of Znchelatase activity in plant extracts (14,15). Therefore, although our buffers contained enough EDTA to sequester contaminating zinc (1,4), it was still prudent to control for Zn-chelatase activity.…”

Section: Resultsmentioning

confidence: 99%

In vitro assay of the chlorophyll biosynthetic enzyme Mg-chelatase: resolution of the activity into soluble and membrane-bound fractions.

Walker

Weinstein

1991

Proc. Natl. Acad. Sci. U.S.A.

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The fist committed step in chlorophyll synthesis is the Mg-chelatase-catalyzed insertion of magnesium into protoporphyrin IX. Since iron insertion into protoporphyrin leads to heme formation, Mg-chelastase lies at the branch point of heme and chlorophyll synthesis in chloroplasts. Little is known about the enzymology or regulation of Mg-chelatase, as it has been assayed only in intact cucumber chloroplasts. In this report we describe an in vitro assay for Mg-chelatase.Mg-chelatase activity in intact pea chloroplasts was 3-to 4-fold higher than in cucumber chloroplasts. This activity survived chloroplast lysis and could be fractionated by centrifugation into supernatant and pellet components. Both of these fractions were required to reconstitute Mg-chelatase activity, and both were inactivated by boiling indicating that the enzyme is composed of soluble and membrane-bound protein(s). The product of the reaction was confirmed fluorometrically as the magnesium chelate of the porphyrin substrate. The specific activity of the reconstituted system was typically 1 nmol of Mg-deuteroporphyrin per h per mg of protein, and activity was linear for at least 60 min under our assay conditions. ATP and magnesium were required for Mg-chelatase activity and the enzyme was sensitive to the sulfhydryl reagent N-ethyhnaleimide (I50, 20 ,uM). Broken and reconstituted cucumber chloroplasts were unable to maintain Mg-chelatase activity. However, the cucumber supernatant fraction was active when combined with the pellet fraction of peas; the converse was not

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Section: Resultsmentioning

confidence: 99%

In vitro assay of the chlorophyll biosynthetic enzyme Mg-chelatase: resolution of the activity into soluble and membrane-bound fractions.

Walker

Weinstein

1991

Proc. Natl. Acad. Sci. U.S.A.

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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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confidence: 99%

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 enzyme catalyzing this insertion is known as magnesium-protoporphyrin IX chelatase and it lies at the branch point of the heme and the bacteriochlorophyll/chlorophyll biosynthetic pathways. Despite the importance of (bacterio)chlorophyll biosynthesis, there is relatively little known about the detailed enzymology and protein chemistry of this pathway, and in the case of Mg chelatase biochemical analyses have been confined to assays using intact cells of the photosynthetic bacterium Rhodobactersphaeroides (1,2), isolated plastids (3)(4)(5)(6), and broken plastid systems (7)(8)(9)(10)(11)(12). In these systems, ATP is absolutely required for magnesium chelatase activity (4).…”

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confidence: 99%

Magnesium-protoporphyrin chelatase of Rhodobacter sphaeroides: reconstitution of activity by combining the products of the bchH, -I, and -D genes expressed in Escherichia coli.

Gibson

Willows

Kannangara

et al. 1995

Proc. Natl. Acad. Sci. U.S.A.

183

165

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Magnesium-protoporphyrin chelatase lies at the branch point of the heme and (bacterio)chlorophyll biosynthetic pathways. In this work, the photosynthetic bacterium Rhodobacter sphaeroides has been used as a model system for the study of this reaction. The Photosynthetic organisms synthesize both chlorophyll and heme, the two major tetrapyrroles in nature. The biosynthetic pathways of these two porphyrins utilize a number of intermediates in common and the first step unique to chlorophyll production is the insertion of Mg into protoporphyrin IX (see Fig. 1). The enzyme catalyzing this insertion is known as magnesium-protoporphyrin IX chelatase and it lies at the branch point of the heme and the bacteriochlorophyll/chlorophyll biosynthetic pathways. Despite the importance of (bacterio)chlorophyll biosynthesis, there is relatively little known about the detailed enzymology and protein chemistry of this pathway, and in the case of Mg chelatase biochemical analyses have been confined to assays using intact cells of the photosynthetic bacterium Rhodobactersphaeroides (1, 2), isolated plastids (3-6), and broken plastid systems (7)(8)(9)(10)(11)(12). In these systems, ATP is absolutely required for magnesium chelatase activity (4). Furthermore, it has been demonstrated with extracts of pea (Pisum sativum) chloroplasts that two components, one soluble and the other with membrane affinity, participate in the enzymatic reaction and that there is an ATP requirement for the activation of these two components (10). Recently, the analysis of this pathway in photosynthetic bacteria has provided a way forward (for a review, see ref. 13). This approach benefits from the availability of the genes for bacteriochlorophyll biosynthesis in Rhodobacter capsulatus and R. sphaeroides, which are clustered on a small region of the genome, -45 kb long (14-17). The gene assignments have been based on the results of insertional mutagenesis, which have been correlated with the accumulation of biosynthetic intermediates, or by the measurement of enzymatic activities (15,(17)(18)(19). Positive identification of function has been lacking, but two recent publications describe the overexpression of the bchM gene from both R. sphaeroides and R. capsulatus in Escherichia coli and demonstrate that the extracts of the E. coli transformants can convert Mg-protoporphyrin IX to Mgprotoporphyrin monomethyl ester (20,21). Apart from positively identifying bchM as the gene encoding the Mgprotoporphyrin methyltransferase, this work opens up the possibility of extending this approach to other parts of the pathway. In this paper, we report the expression of the genes bchH, -I, and -D from R. sphaeroides in E. coli: extracts from these transformants, when combined in vitro, are highly active in catalyzing the chelation of Mg by protoporphyrin IX in an ATP-dependent manner. This is an important step forward since apart from identifying the role of three more bch genes-bchH, -I, and -D-it will allow the biochemistry of this reaction to be studied in deta...

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The synthesis of magnesium and zinc protoporphyrin IX and their monomethyl esters in etioplast preparations studied by high pressure liquid chromatography

Cited by 16 publications

References 15 publications

In vitro assay of the chlorophyll biosynthetic enzyme Mg-chelatase: resolution of the activity into soluble and membrane-bound fractions.

In vitro assay of the chlorophyll biosynthetic enzyme Mg-chelatase: resolution of the activity into soluble and membrane-bound fractions.

Three Separate Proteins Constitute the Magnesium Chelatase of Rhodobacter Sphaeroides

Magnesium-protoporphyrin chelatase of Rhodobacter sphaeroides: reconstitution of activity by combining the products of the bchH, -I, and -D genes expressed in Escherichia coli.

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