Hydrogenation of Geranylgeraniol

Abstract

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.

Kinetics of Chlorophyll Accumulation and Formation of Chlorophyll-Protein Complexes during Greening of Chlamydomonas reinhardtii y-1 at 38°C

“…Instead, the predominant forms during this time were geranylgeraniol Chlides. Thus our results with the algal cells confirm the conclusion reached for etioplasts of higher plants (27,28) that the first pathway described above is used essentially exclusively during the early period of greening.…”

“…Assuming that Chl synthetase in the alga resides in thylakoid membranes, as it does in spinach (28), these results suggest that during greening the final stages of Chl synthesis and assembly of Chlprotein complexes in the alga occur in the residual thylakoid membranes. As further support for this conclusion, most of the protochlorophyll(ide) in dark-grown algal cells is recovered in membranes with the same buoyant density as thylakoid membranes after sucrose gradient separation (5).…”

“…The existence of Chlide b in higher plants (9) and our previously obtained evidence that Chlide b is derived directly from protochlorophyllide (3,4) suggest that during greening the a/b ratio was established prior to esterification. Moreover, the similarities in their kinetic patterns suggest that Chlides a and b enter a common pool and are esterified and reduced by the same enzymatic system (28).…”

“…During greening the initial rates of synthesis and esterification of Chlides with geranylgeranyl pyrophosphate apparently exceed the capacity to reduce the sidechain to the phytol moiety. Soll et al (28) showed that two pathways exist in spinach for generation of the sidechain of Chl. In the first, geranylgeraniol pyrophosphate, synthesized in the chloroplast stroma, is esterified to Chlides by Chl synthetase in thylakoid membranes and then reduced stepwise to produce the phytol-containing Chl.…”

Cloning and Characterisation of Chlorophyll Synthase from Avena sativa

“…In etioplasts and chloroplasts, PhyPP and GGPP gave indistinguishable results when they were applied at saturating concentration and when only the end point of the chlorophyll synthase reaction was analysed. Differences were found when either both substrates were mixed and reacted in competition (Rüdi-ger et al, 1980) or when the kinetics of the reaction was analysed (Soll et al, 1983). Under both conditions, the recombinant enzyme from A. sativa reacts faster with GGPP than with PhyPP at the beginning, indicating a preference for GGPP at short reaction periods.…”