Further Characterization of the Magnesium Chelatase in Isolated Developing Cucumber Chloroplasts

Walker, Caroline J.; Weinstein, Jon D.

doi:10.1104/pp.95.4.1189

Cited by 91 publications

(64 citation statements)

References 25 publications

(48 reference statements)

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“…It was recently shown that pretreatment of seedlings with ␦-aminolevulinic acid causes an increase in the endogenous level of Mg-Proto and Mg-Proto monomethylester, and plastids from these plants had decreased Mg-chelatase levels (Averina et al, 1996). The effective plastid porphyrin concentrations were not given, and we have observed nonspecific inhibition by exogenous porphyrins (Walker and Weinstein, 1991a). Therefore, it is difficult to know if the effect is physiologically important.…”

Section: Discussionmentioning

confidence: 96%

“…When tested on intact plastids from greening cucumber (Cucumus sativus L.) cotyledon, there was no effect on activity from the possible feedback inhibitors protochlorophyllide, Mg-Proto, and chlorophyllide (Walker and Weinstein, 1991a). To ensure that there was not a problem attributable to accessibility caused by the membranes, we retested the effects on activity of the following compounds: Mg-Proto, Mg-Proto monomethylester, and protochlorophyllide.…”

Section: Discussionmentioning

confidence: 99%

“…Mg-Deutero was quantitated on a Perkin-Elmer 650-40 fluorometer calibrated with a Rhodamine block as previously described (Walker and Weinstein, 1991a). Activities are expressed as units (the number of picomoles of MgDeutero produced in a 30-min assay).…”

Section: Stopped Assaymentioning

confidence: 99%

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Magnesium-Chelatase from Developing Pea Leaves1

1998

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Mg-chelatase catalyzes the ATP-dependent insertion of Mg 2؉ into protoporphyrin-IX to form Mg-protoporphyrin-IX. This is the first step unique to chlorophyll synthesis, and it lies at the branch point for porphyrin utilization; the other branch leads to heme. Using the stromal fraction of pea (Pisum sativum L. cv Spring) chloroplasts, we have prepared Mg-chelatase in a highly active (1000 pmol 30 min ؊1 mg ؊1 ) and stable form. The reaction had a lag in the time course, which was overcome by preincubation with ATP. The concentration curves for ATP and Mg 2؉ were sigmoidal, with apparent K m values for Mg 2؉ and ATP of 14.3 and 0.35 mM, respectively. The K m for deuteroporphyrin was 8 nM. This K m is 300 times lower than the published porphyrin K m for ferrochelatase. The soluble extract was separated into three fractions by chromatography on blue agarose, followed by size-selective centrifugal ultrafiltration of the column flow-through. All three fractions were required for activity, clearly demonstrating that the plant Mg-chelatase requires at least three protein components. Additionally, only two of the components were required for activation; both were contained in the flow-through from the blue-agarose column.The biosynthesis of heme and chlorophyll are integral parts of chloroplast development and the acquisition of photosynthetic capacity. Both end products are required in specific but vastly different amounts and are synthesized within the chloroplast via a common tetrapyrrole biosynthetic pathway (Beale and Weinstein, 1990;Porra, 1997). The branch point for the formation of chlorophyll and heme is the use of Proto by the two enzymes that catalyze metal insertion. Ferrochelatase catalyzes the insertion of Fe 2ϩ into Proto, whereas Mg-chelatase catalyzes the insertion of Mg 2ϩ into Proto. Because the flux of common precursors to the respective end products is likely to be controlled by modulation of the branch-point enzymes, we were interested in the properties of these enzymes. This report describes the properties of a totally soluble enzyme system from pea (Pisum sativum L. cv Spring) chloroplasts that catalyzes the Mg-chelatase reaction.Previous work has shown that Mg-chelatase activity requires ATP (Pardo et al., 1980; Walker and Weinstein, 1991b). Although the reaction is formally similar to the ferrochelatase reaction, insertion of a divalent cation into Proto, there is no ATP requirement for the Fe-insertion reaction. Our previous work has shown that the Mgchelatase reaction requires at least two different protein components (Walker and Weinstein, 1991b; Walker et al., 1992). We also showed that the reaction proceeds by a two-step mechanism, involving activation followed by Mg 2ϩ insertion; both steps require ATP (Walker and Weinstein, 1994). This hypothesis was based on the following observations. There is a lag phase in the kinetics that can be overcome by preincubation of the crude enzyme fraction with ATP before the porphyrin substrate is added. ATP␥S can substitute for ATP in the preincubatio...

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Section: Discussionmentioning

confidence: 96%

Section: Discussionmentioning

confidence: 99%

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Magnesium-Chelatase from Developing Pea Leaves1

1998

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“…This is supported by the recent observation that the artificial in i o elevation of Mg-Proto and Mg-Proto monomethyl ester by feeding ALA to pea seedlings caused a 95 % decrease in the initro-measurable Mg-chelatase activity in isolated pea chloroplasts [58]. However, when these intermediates were directly tested in chloroplasts, and more recently in a solubilized Mgchelatase preparation, no evidence for feedback inhibition by Mg-Proto, Mg-Proto monomethyl ester or Pchlide could be found [16,28]. Since these intermediates do not directly inhibit Mg-chelatase, the only explanation for their apparent inhibition in the ALA-feeding experiments is by reducing the amount of one or more of the Mg-chelatase subunits present.…”

Section: Regulation Of Mg-chelatase and Ferrochelatase At The Branch-mentioning

confidence: 93%

“…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

226

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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