Chlorophyll synthetase cannot synthesize chlorophyll a′

Abstract

Chlorophyll synthetase catalyzes the last step of chlorophyll biosynthesis, namely prenylation (esterification) of chlorophyllide with phytyl diphosphate or geranylgeranyl diphosphate. During investigation of various chlorophyllide derivatives as potential substrates we observed lower esterification with increasing percentages of chlorophyllide a' in epimeric mixtures of chlorophyllides a and a: To avoid epimerization during esterification, we studied the reaction in detail with model compounds [zinc-1 32(R)-methoxy-pheophorbide a and zinc-1 3'(S)-rnethoxy-pheophorbide a, zinc-1 32(R)-methoxy-pyropheophorbide a and zinc-chlorin e,-l3', 1 5'-dimethylester]. We conclude that compounds which have the 13'-carbomethoxy group at the same side of the macrocycle as the propionic side chain of ring D are neither substrates nor competitive inhibitors. Only compounds having the 132-carbomethoxy group at the opposite site are substrates for the enzyme. Naturally occuring chlorophyll a ' must be formed by epimerization after esterification.Chlorophyll a' [a-'prime', 13Z(S)-chlorophyll a ] has been known since 1942 (Strain and Manning, 1942) as a byproduct of isolation of chlorophyll a [13'(R)-chlorophyll a]. Due to the easy epimerization of chlorophyll at C-13' (Hynninen, 1991) it is generally believed that it is formed from chlorophyll a during the extraction procedure. However, increasing evidence has accumulated during the last decade that chlorophyll a' is a natural constituent of higher plants and cyanobacteria (Watanabe et al., 1985 a,b;Kobayashi et al., 1988). Investigations on pigment composition of Chlamydomonas reinhardtii (Maroc and Tremolieres, 1990) and of P700-enriched chloroplasts of higher plants (Maeda et al., 1992) revealed that two chlorophyll a' molecules are situated in the core of photosystem I. Furthermore, the presence of two bacteriochlorophyll g 'molecules in the reaction center of heliobacteria was also described (Kobayashi et al., 1990(Kobayashi et al., , 1991. The question now arises, at which stage of the biosynthetic pathway of the chlorophylls is the prime pigment synthesized, especially whether it is formed before or after esterification of chlorophyllide a.Chlorophyll synthetase catalyzes prenylation of chlorophyllides with geranylgeranyl diphosphate (GerGerP,) or phytyl diphosphate (PhyP,), the last step of chlorophyll biosynthesis (Rudiger et al., 1980). This step is essential for translation and accumulation of chlorophyll a apoproteins (Eichacker et al., 1990(Eichacker et al., , 1992 and probably for stable assembly also for other components of the thylakoid membrane (Paulsen et al., 1990; Rudiger 1992 Rudiger , 1993. Chlorophyll synthetase catalyzes prenylation not only of chlorophyllide a, but also of chlorophyllide b and some modified derivatives (Benz and Rudiger, 1981 and Rudiger, 1992;Vezitskii and Sherbakov, 1987). During our studies on the substrate specificity of chlorophyll synthetase, we observed fractions of chlorophyllide a with a greatly reduced ability for esterificat...

Pre-Loading of Chlorophyll Synthase with Tetraprenyl Diphosphate Is an Obligatory Step in Chlorophyll Biosynthesis

“…Preliminary studies of the change of esterification rate with substrate concentration, performed with the method of Helfrich et al (1994), indicated a 'normal' saturation curve for PhyPP ( Figure 3A) and the characteristic of substrate inhibition for Chlide ( Figure 3B) with the optimum reaction rate occurring at 5 -10 µM Chlide. The variation of substrate concentration in the following detailed investigation was therefore limited for Chlide up to about 8 µM, while a range up to 100 µM was used for PhyPP.…”

“…The reaction was stopped with acetone after specified periods, and the resulting pigment mixture was analyzed for total pigment by spectrophotometry and subsequently by HPLC using a fluorescence detector; this fluorescence assay is more sensitive than that using solvent extraction and absorption measurement as described by Helfrich et al (1994). The sensitivity is especially important for the accurate pigment measurement at the beginning of the reaction, when very little esterified pigment is produced.…”

“…The source of the latter was a preparation of inner membranes of oat etioplasts (Helfrich et al, 1994). As substrates, several modified compounds (1 -11) were added, some of which had already been shown to be more or less well accepted as substrates (Helfrich, 1995).…”

Cloning and Characterisation of Chlorophyll Synthase from Avena sativa

“…After incubation for 45 min at 29 °C, the reaction was stopped by addition of 750 µl acetone, and the precipitated protein was removed through centrifugation. After addition of 40 µl 100 mM Na 2 CO 3 , the clear supernatant was used to quantitate the esterified Chl by phase separation as previously described (Helfrich et al, 1994).…”

“…The activity of chlorophyll synthase was originally detected in etioplast membranes of Avena sativa L. , and several investigations were performed with this plant material Schoch et al, 1980;Benz and Rüdiger, 1981a, b;Lütz et al, 1981;Helfrich and Rüdiger, 1992;Helfrich et al, 1994). The enzyme showed a higher activity with geranylgeranyldiphosphate acid residues 109 -115 and 358 -366 of the G4 sequence of A. thaliana (Gaubier et al, 1995) were selected for PCR amplification with suitable primers.…”