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:

Most of the Chl a in nature is formed via the DV and MV carboxylic Chl a biosynthetic routes depicted in Fig. 2. These routes are referred to as carboxylic routes because the tetrapyrrole intermediates between DV Mg-Proto and MV Chl a all have one or two free carboxylic groups. Furthermore most of the reactions in these two routes are heterogeneous. That is the biosynthesis of most of the MV intermediates can proceed via more than one path. This phenomenon is a manifestation of the overall Chl a biosynthetic heterogeneity (Rebeiz et al, 1983, 1994) that permeates the whole Chl a biosynthetic pathway (also see the fully esterified Chl a biosynthetic routes and the Chl b biosynthetic routes). Chl biosynthetic heterogeneity was discovered when it was realized that most of the tetrapyrrole carboxylic and fully esterified pools of plants consist of DV and MV components (Rebeiz, et al, 1983). The biological significance of this phenomenon is becoming more obvious as the Chl biosynthetic pathway is increasingly viewed in the context of the structural and functional heterogeneity of photosynthetic membranes. It has been proposed that in green plants, the multiplicity of Chl a biosynthetic routes, produce different pools of MV Chl a, at different sites of the photosynthetic membranes (Rebeiz et al, 1983, 1994).

Since some of the early biochemical work was done before discovery of the DV and MV Chl biosynthetic heterogeneity, and before development of appropriate analytical methodologies, it is not certain whether the investigated reactions involved only DV or DV + MV tetrapyrroles. To emphasize this ambiguity, the MV and DV connotation will be omitted from the discussion of the early work. In other words, in this context, Mg-Proto would refer either to DV Mg-Proto, to MV Mg-Proto or to a mixture of both. On the other hands, DV and MV Mg-Proto would refer specifically to the DV and MV tetrapyrrole species respectively.

CARBXLC2.gif - 10.2 K

Fig. 2: Reactions of the DV and MV Carboxylic Chl a Biosynthetic Routes Between Mg-Proto and Chl a

The reactions between ALA and DV Proto are shown in more details in Fig. 1. DV= divinyl; MV = monovinyl; Mg-Proto = IX; Mpe = Mg-Proto monomethyl ester; Pchlide a= protochlorophyllide a; Chlide a = Chlorophyllide a; Chl a = chlorophyll a; 4VMPR = [4-vinyl] Mg-Protoporphyrin IX reductase; 4VPideR = [4-vinyl] protochlorophyllide a reductase; 4VCR = [4-vinyl] chlorophyllide a reductase. Arrows joining the DV and MV branches refer to reactions catalyzed by [4-vinyl] reductases. Adapted from Rebeiz et al, 1983, 1994.. The various routes are believed to lead to the formation of MV Chl a, at different sites of the photosynthetic membranes.

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

DV Mg-Proto
MV Mg-Proto

The 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 by crude homogenates of etiolated wheat (Ellsworth and Hervish, 1976). Soon thereafter the enzymatic insertion of Mg into Proto 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 wa 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). The mechanism of Mg insertion into Proto has not been elucidated and will have to await purification of Mg-Proto chelatase to homogeneity. The enzyme is inhibited by metal chelators and sulfhydryl reagents and its specificity toward Mg-proto is not absolute (Walker and Weinstein, 1991b).

1. Metabolism of divinyl (DV) Mg-Proto

DV Mg-Proto

LPPBP\ - 0.0 K

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 the conversion of exogenous DV Mg-Proto to DV Pchlide a in isolated cucumber etiochloroplasts (Tripathy and Rebeiz, 1986). In this heterogeneous system, the conversion of DV Mg-Proto to DV Pchlide a was accompanied by the formation of 12.6 % 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).

2. 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.

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 (Kim and Rebeiz, 1996a). The reaction is catalyzed by [4-vinyl] Mg-Proto reductase (4VMPR). The enzyme was detected in barley etiochloroplasts and appears to be bound to the plastid membranes. Some response to added NADPH was observed. It is very probable that this vinyl reductase is distinct from [4-vinyl] Pchlide a reductase, which converts DV Pchlide a to MV Pchlide a (Tripathy and Rebeiz, 1988) and [4-vinyl] Chlide a reductase, which converts DV Chlide a to MV Chlide a (Parham and Rebeiz, 1992, 1995). 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 exist which act prior to DV Pchlide a and DV Chlide a vinyl reduction, and which are responsible for the accumulation of MV Mg protoporphyrins in plants. It should be pointed that contrary to other MV intermediates which can be formed via more than one path, MV Mg-Proto can only be formed from DV Mg-Proto (Fig. 2). As the starting point of the MV monocarboxylic Chl a biosynthetic route, this enzyme may play an important regulatory role in Chl a biosynthesis.

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

DV Mpe
MV Mpe

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. Mpe also accumulated 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, on the basis of absorbance spectroscopic determinations. When more powerful fluorescence spectroscopic techniques were used to reinvestigate the Mpe pool of plants it was discovered that this pool was chemically heterogeneous and consisted of DV and monovinyl (MV) components (Belanger and Rebeiz, 1982). MV Mpe is now routinely prepared by incubation of etiolated barley leaves with ALA and DPY (Belanger and Rebeiz, 1982).

The metabolic function of Mpe as a precursor of Pchlide a was demonstrated by conversion of exogenous14C- Mpe and unlabeled-Mpe to Pchlide a [the immediate precursor of chlorophyllide (Chlide) a] in organello (Mattheis and Rebeiz, 1977b), using a cell-free system 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).

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).

SAMMT2.gif - 3.5 K

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).

1. 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 determined 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 cucumber etiochloroplasts (Tripathy and Rebeiz, 1986). In this heterogeneous system, conversion of DV Mpe to DV Pchlide a was accompanied by the formation of 20.7 % MV Pchlide a.

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, which may in turn dictate the DV or MV nature of the Mpe substrate, it is not possible to assign with certainty a precise mechanism of action for SAMMT with pure DV Mg-Proto as a substrate.

2. 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). The specific role of MV Mpe as a precursor of one of the MV Pchlide a pools was demonstrated by conversion of exogenous MV Mpe to MV Pchlide a in isolated cucumber and barley etiochloroplasts (Tripathy and Rebeiz, 1986).

MV Mpe can only be formed by methylation of the propionic acid residue of MV Mg-Proto at position 6 of the macrocycle. 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. 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 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 backed-up DV-Mg-Proto and MV Mg-Proto. Whether the SAMMT enzyme that converts MV Mg-Proto to MV Mg-Proto is the same as the one that catalyzes the conversion of DV Mg-Proto to DV Mpe remains to be determined.

References

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