Magnesium-dependent ATPase Activity and Cooperativity of Magnesium Chelatase from Synechocystis sp. PCC6803

Abstract

The first committed step in chlorophyll biosynthesis is catalyzed by magnesium chelatase, a complex enzyme with at least three substrates, cooperative Mg 2؉ activation, and free energy coupling between ATP hydrolysis and metal-ion chelation. A detailed functional study of the behavior of the intact magnesium chelatase has been performed, including characterization of magnesium cooperativity and the stoichiometry of ATP consumption in relation to the magnesium porphyrin produced. It is demonstrated that, in vitro, this catalyzed reaction requires hydrolysis of ϳ15 MgATP 2؊ and that the chelation partial reaction is energetically unfavorable, under our assay conditions, with a ⌬G° of 25-33 kJ mol ؊1 . Given the likely metabolite concentrations in vivo, this results in the chelatase reaction operating far from equilibrium. We have also determined the steadystate kinetic behavior of the intact enzyme and have compared the kinetic parameters obtained with those observed for the partial reactions of individual subunits. K DIX (where D IX represents deuteroporphyrin IX) is estimated to be 3.20 M, and K MgATP 2؊ is 0.45 mM. k cat for chelation is estimated to be 0.8 min ؊1 , suggesting that the ATP hydrolysis catalyzed by the isolated ChlI subunit is substantially slower in the intact chelatase. The magnesium-rich form of the chelatase is a more effective catalyst of the chelation reaction; magnesium activation of the chelatase increases V, as well as the specificity constant for the reaction of MgATP 2؊ and D IX , possibly as a result of a magnesium-triggered conformational change.Chlorophyll is synthesized via a series of tetrapyrrole intermediates; the path toward this pigment diverges from heme biosynthesis at the chelation steps. The chelation step in chlorophyll biosynthesis, catalyzed by magnesium chelatase (E.C. 6.6.1.1), requires ATP hydrolysis and has long been presumed to be energetically unfavorable. The minimum catalytic unit of magnesium chelation requires three proteins, I (38 -42 kDa), D (60 -74 kDa), and H (140 -150 kDa), both in (bacterio)chlorophyll a-producing prokaryotes (1) and in chlorophyll a-synthesizing bacteria and higher plants (2, 3). Studies on purified proteins show that I is the ATPase subunit (4), that it contains a Mg 2ϩ binding site (5), and that it has a fold characteristic of a member of the AAA ϩ superfamily (6). The D subunit forms a stable complex with the I subunit (4) and contains a metal binding motif (6), although no metal binding has yet been demonstrated. The H subunit binds porphyrins (7, 8) and presumably also contains the active site for chelation. As the intact chelatase is a complex enzyme with at least three substrates, cooperative Mg 2ϩ activation, and free energy coupling between ATP hydrolysis and metal-ion chelation, existing mechanistic studies have only provided an overview of the behavior of this enzyme (9, 10).In light of recent studies clarifying the role of individual subunits (4 -8), a detailed functional study of the behavior of the intact magnesium chelata...

The CHLI1 Subunit of Arabidopsis thaliana Magnesium Chelatase Is a Target Protein of the Chloroplast Thioredoxin

“…Thus, magnesium chelatase activity must be primarily controlled by the expression of CHLI and CHLH, a somewhat long range regulatory system; additional post-transcriptional/translational regulation independent from rhythmic regulation is likely to also be involved in the control of the magnesium chelatase in vivo as a short range regulation system. Reid and Hunter (15) reported that the activity of magnesium chelatase is regulated in part by a cooperative response to Mg 2ϩ . Here, we reported that the redox-dependent control of magnesium chelatase constitutes an additional, important regulation mechanism for this enzyme.…”

“…Magnesium chelatase activity is also enhanced by the addition of the GUN4 protein, whose mutation causes a gun phenotype in Arabidopsis (14). (15). In the dark, Mg 2ϩ concentrations in chloroplasts are low, and magnesium chelatase is essentially inactive, whereas the Mg 2ϩ concentrations increase in the light, and the enzymes become active, permitting the flux of the substrate, protoporphyrin IX, into the chlorophyll biosynthetic pathway.…”

Spark plasma sintering of commercial and development titanium alloy powders

“…11 The regulatory enzymology of magnesium chelatase is complex and speciesdependent, with the Synechocystis enzyme showing a complex cooperative response to free magnesium concentration, whereas the enzyme from the thermophile Thermosynechococcus elongatus shows a noncooperative response. 8,12 The magnesium chelatases from these two species have a high degree of sequence identity (82% for ChlI, 67% for ChlD, and 78% for ChlH), and hybrid chelatases composed of subunits from both species are active. Assays of these hybrid chelatases reveal that the Synechocystis ChlD protein is essential for magnesium cooperativity.…”

Abolition of magnesium chelatase activity by thegun5mutation and reversal by Gun4

“…They both carry single point mutations in a conserved region of the H subunit of Mg-chelatase (A990V in gun5-1 and P642L in cch) [3] though there are currently no data pinpointing the effect of these mutations on the Mg-chelatase reaction. Although there is no in vitro assay using purified recombinant chelatase subunits from Arabidopsis, there is a kinetically well-established system for the Synechocystis enzyme [5,11,18].…”