Chlorophyll biosynthesis is a process involving Ϸ20 different enzymatic steps. Half of these steps are common to the biosynthesis of other tetrapyrroles, such as heme. One of the least understood enzymatic steps is formation of the isocyclic ring, which is a characteristic feature of all (bacterio)chlorophyll molecules. In chloroplasts, formation of the isocyclic ring is an aerobic reaction catalyzed by Mg-protoporphyrin IX monomethyl ester cyclase. An in vitro assay for the aerobic cyclase reaction required membranebound and soluble components from the chloroplasts. Extracts from barley (Hordeum vulgare L.) mutants at the Xantha-l and Viridis-k loci showed no cyclase activity. Fractionation of isolated plastids by Percoll gradient centrifugation showed that xantha-l and viridis-k mutants are defective in components associated with chloroplast membranes. The Xantha-l gene, corresponding to Arabidopsis thaliana CHL27, Rubrivivax gelatinosus acsF, Chlamydomonas reinhardtii CRD1, and CTH1 and situated at the short arm of barley chromosome 3 (3H), was cloned, and the mutations in xantha-l 35 , xantha-l 81 , and xantha-l 82 were characterized. This finding connected biochemical and genetic data because it demonstrated that Xantha-l encodes a membrane-bound cyclase subunit. The evidence suggests that the aerobic cyclase requires at least one soluble and two membrane-bound components.barley ͉ acsF ͉ CHL27 ͉ CRD1 ͉ mutant
Mg(2+)-chelatase catalyses the first step unique to chlorophyll synthesis, namely the insertion of Mg2+ into protoporphyrin IX. When pea (Pisum sativum L., cv. Spring) chloroplasts are lysed in a buffer lacking Mg2+ and the thylakoids removed by centrifugation, the remaining mixture of light membranes and soluble proteins (LM/S) has high Mg(2+)-chelatase activity. Several lines of evidence are presented to show that the Mg2+ insertion catalysed by this preparation is a two-step reaction consisting of activation followed by Mg2+ chelation. An activated state of Mg(2+)-chelatase is achieved by preincubating LM/S with ATP. The activated state is observed as the elimination of the approx. 6 min lag in the rate of Mg2+ chelation on addition of the porphyrin substrate. The activity of LM/S assayed at low protein concentrations can be greatly enhanced by preincubating at high protein concentrations (12 mg/ml is optimal). This activation effect requires the presence of both LM and S fractions, as well as ATP. Both steps require ATP, but at different concentrations; the first step is optimal at > 0.5 mM (EC50 = 0.3 mM) and the second step is optimal at 0.3 mM (EC50 < 0.2 mM). ATP in the first step could be replaced by ATP[S]; this analogue could not sustain activity in the second step. This activated state was stable for at least 30 min at room temperature, but chilling of preincubated LM/S on ice for 30 min caused an almost complete loss of the activated state.
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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