Chlorophyll a Biosynthetic Pathway



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IV. The Chl a Carboxylic Biosynthetic Routes: Reactions Between Mg-Protoporphyrin IX (Mg-Proto) and Protochlorophyllide (Pchlide) a:

The Chl carboxylic biosynthetic routes that give rise to most of the Chl a and b in nature are depicted in Figure 1.

A. The Mg-protoporphyrin IX (Mg-Proto) Pool

DV Mg-Proto
MV Mg-Proto

Mg-Proto is the immediate precursor of Mg-proto monomethyl ester (XXIX, XXV). The proposed role of Mg-Proto as an intermediate in the Chl biosynthetic pathway was based on the detection of Mg-Proto in X-ray Chlorella mutants inhibited in their capacity to form Chl (Granick, 1948b). It was conjectured that since the mutants had lost the ability to form Chl but accumulated Mg-Proto, the latter was a logical precursor of Chl. On the basis of absorbance spectroscopic determinations, the accumulated Mg-Proto was assigned a divinyl (DV) chemical structure. When more powerful fluorescence spectroscopic techniques were used to reinvestigate the Mg-Proto pool of plants it was discovered that this pool was chemically heterogeneous and consisted of DV and monovinyl (MV) components (Belanger and Rebeiz, 1982).

The metabolic role of Mg-Proto as a precursor of other Mg-porphyrins and of Pchlide a was demonstrated by conversion of 3H-Mg-Proto to 3H-Pchlide a (the immediate precursor of chlorophyllide (Chlide) a) by crude homogenates of etiolated wheat (Ellsworth and Hervish, 1976).

1. Mg-Protoporphyrin IX Chelatase
The enzymatic insertion of Mg into Proto by Mg-Proto chelatase, to yield Mg-Proto was achieved in organello (Smith and Rebeiz, 1977a). At the low ATP concentrations used in this system, the biosynthesis of Mg-Proto was accompanied by the formation of Zn-Proto. Simultaneous equations were used in order to deconvolute the fluorescence spectra and be able to determine the amounts of Mg-Proto in the presence of Zn-Proto contamination (Smith and Rebeiz, 1977b). Later on, interference from Zn-Proto was eliminated when it was realized that ATP was a mandatory cofactor for Mg-insertion into Proto and that higher concentration of added ATP eliminated the Zn-problem (Pardo et al, 1980).

In cucumber etiochloroplasts, Mg-Proto chelatase is bound to the plastid membranes (Smith and Rebeiz, 1979), (Lee et al , 1992). The activity of the membrane-bound enzyme increased upon addition of exogenous Mg (Lee et al, 1992). In pea chloroplasts, contrary to what was observed in cucumber plastids, both stroma and membranes were needed to reconstitute Mg-Proto chelatase activity (Walker and Weinstein, 1991a). It is not known whether the discrepancy between the cucumber and pea results are due to differences in preparatory methodologies or not. Indeed it has been reported that the separation of plastid stroma from plastid membranes may result in the solubilization of membrane components if appropriate precautions are overlooked (Lee et al, 1991). In cucumber but not in pea, Mg-Proto chelatase was stabilized by its substrate (exogenous Proto) before separation of the stroma from the plastid membranes (Lee et al, 1992).

Mutational analysis of the Rb. Capsulatus photosynthesis gene cluster suggested that three sequenced genes, namely bchH, bchD and bchI were involved in Mg-chelation (Suzuki et al, 1997). The bchH, bchI and bchD genes from R. spheroides were expressed in E. coli. When cell-free extracts from strains containing the gene products BchH, BchI, and BchD were combined, the mixture was able to catalyze the insertion of Mg into Proto in an ATP-dependent manner (Gibson et al, 1995). The authors suggested that BchH binds Proto prior to the insertion of the Mg atom. Genes from Synechocystis PCC6803 a cyanobacterium, with homology to the bchH, bchD and bchI genes, namely chlH, chlD and chlI, were cloned and overexpressed in E. coli (Jensen et al, 1995). In this case too, the combined cell-free extracts containing the ChlH, ChlI and ChlD gene products were able to catalyze the insertion of Mg2+ into Proto in an ATP-dependent manner. The N-terminal half bof the ChlD protein exhibited a 40-41% homology to Rhodobacter BchI and Synechocystis ChlI, whereas the C-terminal half displayed a 33% homology to Rhodobacter BchD. The authors suggested the existence of an evolutionary relationship between the I and D genes. It is now acknowledged that insertion of Mg2+ into Proto appears to be a two–step reaction, consisting of activation followed by Mg2+ insertion (Jensen et al, 1999). The activation step requires ATP and the ChlI and ChlD subunits and results in the formation of an ChlI-ChlD-ATP complex. Insertion of Mg2+ into Proto also requires ATP and the ChlH subunit. It was observed however that during formation of the ChlI-ChlD-ATP complex, ATP may be replaced by a slowly hydrolysable analog such as 5’-[?-thio]triphosphate, by a non-hydrolysable ATP analog such as adenosine 5;-[?,?-imido] triphosphate, or to a lesser extent by ADP. There was an absolute requirement, however for ATP hydrolysis during Mg2+ insertion by the ChlH protein.

In Arabidopsis thaliana, the ChlH gene product was observed to undergo a dramatic diurnal variation, rising almost to its maximum level by the end of the dark period, increasing slightly at the onset of the light period and declining steadily to a minimum by the end of the light period (Gibson et al, 1996). It was proposed that the ChlH protein plays a role in regulating the levels of chlorophyll during the daily dark-light cycle. Furthermore immunoblotting showed that the distribution of the ChlH protein between the stroma and chloroplast membranes varied depending on the concentration of Mg2+. For example in soybean, the ChlH protein was found either in the stroma at low Mg2+ concentration in the lysing buffer, or on the chloroplast envelope at high lysing buffer Mg2+ concentration (Nakayama et al, 1998)[96].

2. Biosynthetic Heterogeneity of the Mg-Proto Pools

Molecular biological studies of Mg-Proto chelatase (vide supra) have not yet addressed the problem of the spatial and chemical heterogeneities of Mg-Proto formation. Indeed, Mg-Proto is a chemically heterogeneous pool made up of DV and MV components (Belanger and Rebeiz, 1982). The proportion of DV to MV Mg-Proto biosynthesis depends on the greening group affiliation, plant species and pretreatment of plant tissues. For example cucumber cotyledons a dark-divinyl- light-divinyl-light-dark divinyl (DDV-LDV-LDDV) plant tissue (Abd-El-Magid, 1997) , pretreated with 2,2’-dipyridyl (Dpy) accumulate more DV than MV Mg-Proto in darkness. On the other hands, the opposite is true in dark-monovinyl-light-divinyl-light-dark-monovinyl (DMV-LDV-LDMV) plants such as etiolated corn or barley.

In Fig. 1, five DV Mg-Proto and two MV Mg-Proto pools are depicted in seven different thylakoid locations. The assignment of seven Mg-Proto pools to seven different thylakoid environments is based (a) on the detection of multiple resonance excitation transfer bands between Mp(e) [i. e. Mg-Proto + Mg-Proto monomethyl ester (Mpe)] and various Chl-protein complexes (Table 1), and the detection of MV Mg-proto (XXI) in green plants (Belanger and Rebeiz, 1982), and considerations related to the biosynthesis of DV and MV Pchlide a

a. metabolism of divinyl (dv) mg-proto

DV Mg-Proto

dvmp2.gif - 13255 Bytes

The DV nature of the DV Mg-Proto component of the Mg-Proto pool of higher plants was determined by chemical derivatization coupled to analytical fluorescence spectroscopy at 77 K (Belanger and Rebeiz, 1982a).

The specific role of DV Mg-Proto as a precursor of DV Pchlide a was demonstrated by conversion of exogenous DV Mg-Proto to DV Pchlide a in isolated etioplasts of cucumber, a DDV-LDV-LDDV plant species, and barley, a DMV-LDV-LDMV plant species (Abd-El-Magid et al, 1997; Tripathy and Rebeiz, 1986). In cucumber etioplasts, DV Mg-Proto was converted into 89% DV Pchlide a and 11% MV Pchlide a. In barley etioplasts the MV and DV proportions amounted to 16% DV Pchlide a and 84% MV Pchlide a. In both greening groups, the results provided a clear indication that both DV and MV routes were involved in the conversion of DV Mg-Proto to DV and MV Pchlide a.

It has recently become apparent that the biosynthetic heterogeneity of the carboxylic Chl a biosynthetic routes originates in the DV Mg-Proto pool (Kim and Rebeiz, 1996a). With the development of improved techniques for the extraction and determination of DV and MV Proto (Kim and Rebeiz, 1996b), it was shown that under no circumstances was it possible to induce the formation of MV Proto in higher plants tissues. However the conversion of exogenous DV Mg-Proto to MV Mg-Proto in organello (Kim and Rebeiz, 1996a) was readily achieved. This led to the conclusion that the first committed step of the MV carboxylic Chl a biosynthetic route starts with the conversion of DV Mg-Proto to MV Mg-Proto. The specific biosynthesis of DV Mg-Proto from DV Proto was first reported in cucumber etiochloroplasts in the presence of added ATP and Mg (Tripathy and Rebeiz, 1986).

In Fig. 1, five DV Mg-Proto pools are depicted as being formed from DV Proto via routes 1, 8, 10, 11 and 12. At this stage it is unclear whether the spatial biosynthetic heterogeneity of DV Mg-Proto is accompanied by chemical biosynthetic heterogeneity or not. In other words, it is unclear whether the proposed biosyntheses of DV Mg-Proto from DV Proto via routes 1, 8, 10, 11 and 12 are catalyzed by identical Mg-Proto chelatases or by different Mg-Proto chelatase isozymes.

b. Metabolism of monovinyl (MV) Mg-Proto

MV Mg-Proto

mvmp2.gif - 13.4 K

The MV nature of the MV Mg-Proto component of the Mg-Proto pool of higher plants was determined by chemical derivatization coupled to analytical fluorescence spectroscopy at 77 K (Belanger and Rebeiz, 1982a). The specific role of MV Mg-Proto as a precursor of MV Pchlide a was demonstrated by conversion of exogenous MV Mg-Proto to MV Pchlide a in isolated cucumber and barley etiochloroplasts (Tripathy and Rebeiz, 1986). Conversion of MV Mg-Proto to MV Pchlide a was not accompanied by formation of DV Pchlide a.

The MV nature of the MV Mg-Proto component of the Mg-Proto pool was determined by chemical derivatization coupled to analytical fluorescence spectroscopy at 77 °K (Belanger and Rebeiz, 1982).

MV Mg-Proto is formed from DV Mg-Proto by reduction of the vinyl group to ethyl at position 4 (ring B) of the macrocycle (Fig. 1, route 2 and 12) (Kim and Rebeiz, 1996a). The reaction is catalyzed by a putative [4-vinyl] Mg-Proto reductase (4VMPR). This enzyme was detected in barley etiochloroplasts and appears to be bound to the plastid membranes. Its presence in DDV-LDV-LDDV plants as in cucumber cotyledons is strongly suggested by the biosynthesis and accumulation of MV Mg-proto during incubation of etiolated cucumber cotyledons with ALA and Dpy (Belanger and Rebeiz, 1982). A positive response of 4VMPR to added NADPH has been observed (Kim and Rebeiz, 1996).

It is very probable that 4VMPR is distinct from [4-vinyl] Pchlide a reductase (4PideR), which converts DV Pchlide a to MV Pchlide a (Tripathy and Rebeiz, 1988); from [4-vinyl] Chlide a reductase (4VCR), which converts DV Chlide a to MV Chlide a (Parham and Rebeiz, 1992; 1995; Kolossov and Rebeiz, 2001), and from [4-vinyl] Chl a reductase (VChlR) (Adra and Rebeiz, 1998). For example, Rhodobacter capsulatus in which the bchJ gene which codes for DV Pchlide a reductase, has been deleted, accumulates massive amounts of MV Mg-Proto and its monoester (precursors of Pchlide a) in addition to the accumulation of DV Pchlide a, (Suzuki and Bauer, 1995). This in turn indicates that separate [4-vinyl] reductases are active before DV Pchlide a , DV Chlide a and DV Chl a vinyl reduction at position 4 of the macrocycle. It should be pointed that contrary to other MV intermediates which can be formed via more than one biosynthetic route, MV Mg-Proto can only be formed from DV Mg-Proto via routes 2 and 12 ( Fig. 1. It is unknown whether MV Mg-Proto can also be formed by vinyl reduction of DV Mg-Proto formed via routes 8, 10, and 11, (Fig. 1.)

The specific role of MV Mg-Proto as a precursor of MV Pchlide a was demonstrated by conversion of exogenous MV Mg-Proto to MV Pchlide a in isolated cucumber and barley etiochloroplasts (Tripathy and Rebeiz, 1986). Conversion of MV Mg-Proto to MV Pchlide a was not accompanied by formation of DV Pchlide a. This in turn indicates that at the level of MV Mg-Proto, further metabolism can only proceed via an exclusive MV biosynthetic route. In route 12 (Fig. 1), the MV Mg-proto pool is depicted as the immediate precursors of the lone MV Mpe pool.

B. The Mg-Proto monomethyl ester (Mpe) Pool

DV Mpe
MV Mpe

Protoporphyrin IX monomethyl ester (Mpe) is the precursor of Pchlide a (Fig. 1.). The role of Mg-Proto monomethyl ester (Mpe) as an intermediate in the Chl biosynthetic pathway was based on the detection of Mpe in X-ray Chlorella mutants inhibited in their capacity to form Chl (Granick, 1961). It was conjectured that since the mutants had lost the ability to form Chl but accumulated Mpe, the latter was a logical precursor of Chl. On the basis of absorbance spectroscopic analysis, Mpe was assigned a DV chemical structure. Mpe was also detected in barley leaves incubated with ALA and 2,2'-dipyridyl (Dpy) (Granick, 1961). In this case too, the accumulated Mpe was assigned a divinyl (DV) chemical structure. When more powerful fluorescence spectroscopic techniques were used to reinvestigate the chemical nature of Mpe pool of plants it was found to be chemically heterogeneous and to consist of DV and monovinyl (MV) components (Belanger and Rebeiz, 1982). Substrate amounts of MV Mpe are now routinely prepared by incubation of etiolated barley leaves with ALA and Dpy (Rebeiz, 2001). The metabolic function of Mpe as a precursor of Pchlide a was demonstrated by conversion of exogenous [14C]Mpe and unlabeled-Mpe to [14C]Pchlide a, the immediate precursor of Chlide a, in organello (Mattheis and Rebeiz, 1977). In this undertaking, a cell-free system was used, capable of the conversion of 14C-ALA to 14C- Pchlide a, 14C-Pchlide ester a and 14C-Chl a and b (Rebeiz, and Castelfranco, 1971a, Rebeiz and Castelfranco, 1971b), and capable of the net conversion of exogenous ALA to Mg-Protoporphyrins and Pchlide a (Rebeiz, et al, 1975).

1. (-) S-Adenosyl-L-Methionine-Magnesium Protoporphyrin Methyl Transferase

sammt2.gif - 3.5 K

Mg-Proto is converted to Mpe by transfer of a methyl group from (-) S-adenosinyl-L- methionine (SAM) to Mg-Proto. the reaction results in the methyl esterification of the propionic acid residue at position 6 (ring C) of the macrocycle. The reaction is catalyzed by (-) S-adenosyl-L-methionine-magnesium protoporphyrin methyl transferase (SAMMT).

The occurrence of SAMMT was first reported in Rhodopseudomonas spheroides (Gibson et al, 1963). The enzyme was confined to the chromatophores to which it was firmly bound. Substrate specificity was lax since in addition to Mg-Proto, zinc proto, calcium Proto, Mg-mesoporphyrin and Mg-deuteroporphyrin also acted as substrates. S-adenosyl homocysteine and S-adenosylethionine inhibited the reaction competitively. The enzyme has also been detected in corn (Zea mays) chloroplasts (Radmer and Bogorad, 1967). A 1600-fold purification of the R. spheroides enzyme was achieved by affinity chromatography (Hinchigeri et al, 1984). The purified enzyme exhibited an equilibrium-ordered sequential Bi Bi mechanism with Mg-Proto as the obligatory first substrate, and SAM as the second substrate. The nucleotide sequence of the R. capsulatus enzyme has been reported (Bollivar and Bauer, 1992).

Originally, in R. capsulatus, SAMMT was believed to be coded for by the bchH gene, while the bchM gene was believed to code for a polypeptide involved in the formation of the cyclopentanone ring (ring E) of Pchlide a (Bauer et al, 1993). Later on, the bchM gene of R. capsulatus was expressed in E. coli and the gene product was subsequently demonstrated by enzymatic analysis to catalyze methylation of Mg-proto to form Mpe (Bollivar et al, 1994). Activity required the substrates Mg-proto and S-adenosyl-L-methionine. To our knowledge, no higher plant SAMMT gene has been isolated. A query for SAMMT addressed to the various protein databases listed in the Biology Workbench, yielded 3 unique records which are depicted on the vLPBP website at “http://www.vlpbp.org/greening/XVI. Sequenced Enzymes/ SAM-Mg-proto MT”. These sequences can be viewed and used for sequence similarity searches or other manipulations using the Biology Workbench.

2. Biosynthetic Heterogeneity of the Mg-Proto Monomethyl Ester Pool (Mpe)

Mpe is a chemically heterogeneous pool made up of DV and MV components (Belanger and Rebeiz, 1982). As was observed for Mg-Proto, the proportion of DV to MV Mg-Proto biosynthesis depends on the greening group affiliation, plant species and pretreatment of plant tissues. For example cucumber cotyledons a DDV-LDV-LDDV plant tissue (Abd-El-Magid et al, 1997), pretreated with Dpy accumulate more DV than MV Mpe in darkness. On the other hands, the opposite is true in DMV-LDV-LDMV tissues such as etiolated corn or barley leaves.

In Fig. 1, five DV Mpe and two MV Mpe pools are depicted in seven different thylakoid environments. The assignment of seven Mpe pools to seven different thylakoid locations is based on the detection of multiple resonance excitation transfer bands between Mp(e) and various Chl-protein complexes (Table1), the detection of MV Mpe in plants (Belanger and Rebeiz, 1982), and considerations related to the biosynthesis of DV and MV Pchlide a.

a. Metabolism of DV Mpe

DV Mpe

dvmpe2.gif - 16.3 K

The DV nature of the DV Mpe component of the Mpe pool of higher plants was confirmed by chemical derivatization coupled to analytical fluorescence spectroscopy at 77 K (Belanger and Rebeiz, 1982a).

The specific role of DV Mpe as a precursor of DV Pchlide a was demonstrated by conversion of exogenous DV Mpe to DV Pchlide a in isolated etiochloroplasts of cucumber, a DDV-LDV-LDDV plant species, and barley, a DMV-LDV-LDMV plant species (Abd-El Mageed et al, 1997) Tripathy and Rebeiz, 1986). In cucumber etioplasts, DV Mpe was converted into 83% DV Pchlide a and 17% MV Pchlide a. In barley etioplasts, DV Mpe was converted into 44% DV Pchlide a and 56% MV Pchlide a. The results provided a clear indication that both DV and MV routes were involved in the conversion of DV Mpe to DV and MV Pchlide a in both greening groups.

To our knowledge, no kinetic studies have been performed on SAMMT purified to homogeneity, using pure DV Mpe. Since the mechanism of action of SAMMT has been reported to vary i. e. ping pong (Ellsworth et al, 1974), random Bi Bi (Ebbon and Tate, 1969), or ordered Bi Bi (Hinchigeri et al, 1984) depending on the source of enzyme, it is not possible to assign with certainty a precise mechanism for its action without precise knowledge of the DV or MV nature of the Mpe substrate.

In Fig. 1, the five DV Mpe pools are depicted as being formed from DV Mg-Proto via routes 1, 8, 10, 11 and 13. At this stage it is unclear whether the spatial biosynthetic heterogeneity indicated by multiple resonance excitation energy transfer bands (Table 1) is accompanied by chemical biosynthetic heterogeneity or not. In other words, it is unclear whether the biosynthesis of DV Mpe from DV Mg-Proto via routes 1, 8, 10, 11 and 13 is catalyzed by identical SAMMTs or by SAMMT isozymes.

b. Metabolism of MV Mpe

MV Mpe

mvmpe2.gif - 16.9 K

The MV nature of the MV Mpe component of the Mpe pool of higher plants was determined by chemical derivatization coupled to analytical fluorescence spectroscopy at 77 K (Belanger and Rebeiz, 1982a). MV Mpe can only be formed by methylation of the propionic acid residue of MV Mg-Proto at position 6 of the macrocycle. Indeed, contrary to previous claims, conversion of DV Mpe to MV Mpe (Ellsworth and Hsing, 1973) by a [4-vinyl] Mpe reductase, could not be demonstrated in higher plants, under conditions that led to the ready conversion of DV Mg-Proto to MV Mg-Proto (Kim and Rebeiz, 1996a). In other words, esterification of MV Mg-Proto to MV Mpe appears to be the primary regulatory reaction in the biosynthesis of MV Mpe. In Rhodobacter capsulatus from which the bchJ gene which codes for DV Pchlide a reductase, has been deleted, accumulates massive amounts of MV Mg-Proto and MV Mpe, in addition to the accumulation of DV Pchlide a, (Suzuki and Bauer, 1995). Accumulation of MV Mpe in this system probably proceeds via methyl esterification of MV Mg-Proto. Whether the SAMMT enzyme that converts MV Mg-Proto to MV Mpe is the same as the one that catalyzes the conversion of DV Mg-Proto to DV Mpe remains to be determined.

The specific role of MV Mpe as a precursor of one of two of the MV Pchlide a pools was demonstrated by exclusive conversion of exogenous MV Mpe to MV Pchlide a in isolated cucumber and barley etiochloroplasts (Tripathy and Rebeiz, 1986). This in turn indicates that at the level of MV Mpe, further metabolism can only proceed via exclusive MV biosynthetic routes. In biosynthetic routes 2 and 12 (Fig. 1), the MV Mpe pool is depicted as the immediate precursors of two MV Pchlide a pools.

C. The Mg-Proto Diester (Mpde) Pool

Mg-proto diesters (Mpdes) (XXVI, XXVII) are the first metabolic intermediates of the fully esterified Chl a biosynthetic routes (Fig. 2, routes 16, 17). The fully esterified Chl a pathway is populated by tetrapyrroles with a methyl propionate residue at position 6 of the macrocycle and a propionic acid residue at position 7 which is esterified with one of several different long chain fatty alcohols (LCFAs) [17]. The fully esterified biosynthetic routes of the Chl a biosynthetic pathway deal with the least understood phases of the intermediary metabolism of Chl a. In our opinion, the unjustified neglect of this facet of Chl a biosynthesis is motivated by several factors, among which (a) occurrence of metabolic intermediates in very small amounts, (b) slow reaction rates, (c) analytical difficulties, and (d) misconceptions that date back to the late 1930s (see section 12) A fully esterified Mpde pool was first detected in etiolated cucumber cotyledons incubated overnight with ALA and Dpy in darkness [115]. The novel pool exhibited the chromatographic properties of a fully esterified metalloporphyrin and the spectrofluorometric properties of Mg-Proto. Chemical derivatization coupled to spectrofluorometric and chromatographic analysis identified it as a Mg-Proto diester (Mpde). Mpde was also detected in dark-grown Euglena gracilis and in etiolated cucumber cotyledons incubated in darkness with ALA, in the absence of added Dpy. Upon detection of Mpde, it was suggested to be a metabolic precursor of the fully esterified, heterogeneous, Pchlide a ester pool [115].
1. Biosynthetic Heterogeneity of the Mg-Proto Diester Pool
High-pressure liquid chromatographic analysis indicated that the Mpde pool was heterogeneous and consisted of three fully esterified Mg-Protos. Gas-chromatographic /mass spectroscopic analysis of the saponified alcohol fraction of the heterogeneous Mpde pool revealed that the latter consisted of three major long-chain alcohols, none of which was identifiable with known isoprenoids such as, farnesol or phytol [115]. In addition to the heterogeneity of the long chain alcohols esterifying the propionic acid residue at position 7 of the macrocycle, the Mpde pool exhibited a well pronounced DV-MV chemical heterogeneity [83]. For example in etiolated cucumber cotyledons incubated in darkness with ALA and Dpy, as well as in dark-grown Euglena gracilis, the Mpde pool consisted of DV and MV Mpde components. In general the proportion of DV Mpde is higher than that of MV Mpde, except in Euglena.
a. Metabolism of DV Mpde
The biosynthetic origin of DV Mpde is not presently clear, and is tentatively assigned to DV Mpe esterification (Fig. 2, route 17). After its detection in cucumber cotyledons and in Euglena cultures [83], DV Mpde (XXVI), has been proposed as a precursor of fully esterified DV Pchlide a, i. e. DV Pchlide a ester (DV Pchlide a E) (Fig. 2, route 17). A precursor-product relationship between DV Mpde and DV Pchlide a E remains to be established and is complicated by the presence of Mpde esterases that convert exogenous Mpde to Mpe (Rebeiz, unpublished).
b. Metabolism of MV Mpde

The biosynthetic origin of MV Mpde(XXVI) is not presently clear, and is tentatively assigned to MV Mpe esterification (Fig. 2, route 16). After its detection in cucumber cotyledons and in Euglena cultures where it is the main constituent of the Mpde pool [83], MV Mpde has been proposed as a precursor of MV Pchlide a E (Fig. 2, route 16). A precursor-product relationship between MV Mpde and DV Pchlide a E remains to be established and is complicated by the presence of Mpde esterases that convert exogenous Mpde to Mpe (Rebeiz, unpublished).

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