CHL27, the Arabidopsis homologue to Chlamydomonas Crd1, a plastid-localized putative diiron protein, is required for the synthesis of protochlorophyllide and therefore is a candidate subunit of the aerobic cyclase in chlorophyll biosynthesis. ␦-Aminolevulinic acid-fed antisense Arabidopsis plants with reduced amounts of Crd1͞CHL27 accumulate Mg-protoporphyrin IX monomethyl ester, the substrate of the cyclase reaction. Mutant plants have chlorotic leaves with reduced abundance of all chlorophyll proteins. Fractionation of Arabidopsis chloroplast membranes shows that Crd1͞ CHL27 is equally distributed on a membrane-weight basis in the thylakoid and inner-envelope membranes.T he chlorophyll (Chl) biosynthetic pathway, occurring in all photosynthetic organisms, has been described through genetic analysis of bacterial mutants and in vitro reconstitution of individual reactions (1-3). Chl production begins with the condensation of eight molecules of ␦-aminolevulinic acid (ALA) to uroporphyrinogen III, the first cyclic tetrapyrrole. Uroporphyrinogen III is then converted to protoporphyrin IX, which is the branch-point intermediate to hemes and Chls. The chelation of magnesium into protoporphyrin IX results in the formation of Mg-protoporphyrin IX (MgP), which is converted to MgP monomethyl ester (MgPMME) by a methyl transferase (4). MgPMME is the substrate for the so-called cyclase reaction, which results in the formation of divinyl protochlorophyllide (Pchlide) containing the fifth ring (ring E) characteristic of all Chls (Fig. 1). In angiosperms, the subsequent steps include the extensively studied, light-dependent conversion of Pchlide to chlorophyllide a via NADPH-Pchlide oxidoreductase and the addition of a polyisoprene tail to complete Chl a production.Labeling experiments (5) suggested two different mechanisms for the cyclase reaction and presumably two different enzymes. The bchE gene product is implicated in the anaerobic reaction because bchE mutants in Rhodobacter sphaeroides accumulate pigments corresponding to MgP and MgPMME (6). The aerobic enzyme is clearly distinct. Although its genetic identity remained elusive for a long time, biochemical studies did define a reaction path and several key features of the enzyme. The aerobic cyclase reaction, an overall six-electron oxidation, is proposed to occur in three sequential steps (7) (Fig. 1). The first step is the stereospecific hydroxylation of the methyl-esterified ring C propionate followed by oxidation of the alcohol to the corresponding ketone. The now-activated methylene group reacts with the ␥-meso carbon of the porphyrin nucleus in an oxidative reaction involving removal of two H to yield ring E. Both the hydroxylated and the keto compounds were suggested to be genuine intermediates, because they could function as substrates for the enzyme (8, 9). Molecular oxygen is required at the step of hydroxylation and also for the conversion of the keto intermediate to divinyl Pchlide (8).The enzyme is iron-dependent: iron chelators inhibit the cyclase enzyme ...
Chloroplast DNA (cpDNA) binds to the envelope membrane of actively dividing chloroplasts (plastids) in young pea leaves. South‐western blotting was used to identify and characterize the protein involved in the binding of cpDNA to the envelope membrane. A 130 kDa protein in the inner chloroplast (plastid) envelope membrane binds specific sequences within the cpDNA. These included a 0.41 kbp sequence located upstream of the psaAB gene, a 0.57 kbp sequence located downstream of the petA gene and a 1.2 kbp sequence located within the rpoC2 gene. The protein was detected in the envelope membrane of young pea leaves in which the cpDNA had been located by fluorescence microscopy at the chloroplast periphery, whereas it was undetectable in mature leaves. We therefore propose that the 130 kDa protein is involved in the binding of cpDNA to the envelope membrane, and named it plastid envelope DNA‐binding protein.
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