The esterification of chlorophyllide a was investigated in irradiated etioplast-membrane fractions ('broken etioplasts') from oat seedlings (Avcnu sativa L.). As a substrate, [l-3H]geranylgeranio1 and its monophosphate and diphosphate derivatives were prepared by chemical synthesis. Geranylgeraniol and its monophosphate derivative are incorporated into chlorophyll only in the presence of ATP whereas the diphosphate derivative is incorporated also without ATP. The yield of esterified chlorophyll is 80 -90 of chlorophyllide with saturating substrate concentrations. The term 'chlorophyll synthetase' is used to describe this enzyme activity which is different from chlorophyllase. Other substrates are phytol and farnesol either with ATP or as the diphosphate derivatives. The relative specificity of 'chlorophyll synthetase' for these substrates is geranylgeraniol : phytol : farnesol = 6 : 3 : 1 .In these experiments in vitro, a new chlorophyll esterified with farnesol was detected which does not occur in intact plants. Geraniol and n-pentadecanol are no substrates fhr the enzyme. Protochlorophyllide which is present in non-irradiated etioplast membrane fractions is not esterified under the same conditions.

The reduction of geranylgeranylpyrophosphate to phytylpyrophosphate in spinach chloroplasts is described for the first time. The reductase is localized in the chloroplast envelope. By Refs. 8,23,22,5,and 24, respectively). GG is known to occur as the side chain in bacteriochlorophyll of purple bacteria (7), and it was later found in trace amounts esterified to Chl a in greening etiolated tissue (12,25). Rudiger et al. (17) showed that an enzyme different from chlorophyllase, i.e., Chl synthetase, was responsible for the esterification of Chlide in etiolated tissue. Subsequently, this enzyme was demonstrated in spinach chloroplasts (6,20) and Chl-free chromoplasts from daffodil (1 1). Further examination of the last steps ofChl biosynthesis in greening etiolated oat seedlings showed a stepwise reduction of the initial product, ChiGG, via Chl dihydrogeranylgeraniol and Chl tetrahydrogeranylgeraniol to Chiph (2,18). In a preliminary report (20), we showed that this sequence also exists in the natural green system. Very little is known about the regulation of this important final step in Chl synthesis in chloroplasts.In studies of tocopherol and phylloquinone synthesis, we found that GGPP could not substitute for PPP in the enzymic prenylation of the aromatic precursor of either vitamin (19,21). This is in contrast to Chl synthetase which uses GGPP and PPP and to a ' This work was aided by grants from the Deutsche Forschungsgemeinschaft.2Abbreviations: Ph, phytol; GG, geranylgeraniol; IPP, isopentenylpyrophosphate; ChlGG.DHGG,THGG,Ph, Chi esterified with GG, dihydro-GG, tetrahydro-GG, and Ph; GGPP and PPP, GG-and Ph-pyrophosphate; Phae, phaeophytin; FPP, farnesol-PP; Chl aF, Chl afarnesoi.lesser extent even FPP in the etioplast system (17). In a recent short communication (20), we showed for the first time that GGPP was directly incorporated into Ph. No data were available for the localization and regulation of this reductase activity. The data presented below further characterize the reduction of GGPP and show that two distinct sites and two distinct pathways exist for the conversion of the geranylgeranyl moiety to the phytyl moiety in spinach chloroplasts: the chloroplast envelope for the hydrogenation of GGPP to PPP, and the thylakoids for the esterification of chlide with GGPP and stepwise reduction of Chlc0 to ChlPh. MATERIALS AND METHODSReagents. The reagents were from commercial sources and were of the highest purity available.

The esterification of various chlorophyllides with geranylgeranyl diphosphate was investigated as catalyzed by the enzyme chlorophyll synthetase. The enzyme source was an etioplast membrane fraction from etiolated oat seedlings (Avena sativa L.). The following chlorophyllides were prepared from the corresponding chlorophylls by the chlorophyllase reaction: chlorophyllide a (2) and b (4), bacteriochlorophyllide a (5), 3-acetyl-3-devinylchlorophyllide a (6), and pyrochlorophyllide a (7). The substrates were solubilized with cholate which reproducibly reduced the activity of chlorophyll synthetase by 40-50% . It was found that the following compounds were good substrates for chlorophyll synthetase: chlorophyllide a and b, 3-acetyl-3-devinylchloro-phyllide a, and pyrochlorophyllide a. Only a poor or no reaction was found with protochloro­phyllide, pheophorbide a, and bacteriochlorophyllide. This difference of reactivity was not due to distribution differences of the substrates between solution and pelletable membrane fraction. Furthermore, no interference between good and poor substrate was detected. Structural features necessary for chlorophyll synthetase substrates were discussed.

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