Chlorophyll b Biosynthetic Pathway

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  • X. The Chl b Biosynthetic Pathway: Intermediary Metabolism

  • A. Determination of Precursor-Product Relationships In Vivo

  • B. Source of Oxygen During the Formation of the Formyl group of Chl b

  • C. References

    X. The Chl b Biosynthetic Pathway


    Fig. 1. Integrated Chl a/b biosynthetic pathway depicting 15 carboxylic routes. To facilitate understanding of the text, various biosynthetic routes are designated by the numbers 1-14. Black boxes with yellow lettering joined by black arrows and numbers refer to biosynthetic routes that occur in etiolated tissues in darkness, or in greening tissues at the beginning of the light phase of the photoperiod. White boxes with green lettering joined by yllowish-brown arrows and numbers refer to biosynthetic routes that occur in green tissues, during the light phases of the photoperiod. Blue frames refer to routes that occurin DDV-LDDV greening groups. Red frames refer to routes that occurin DMV-LDMV greening groups.

    FourteenRoutesCarboxylicPW.jpg - 86601 Bytes


    The demonstration of metabolic pathways is a multistep process. It involves at least three stages: (a) the detection and characterization of metabolic intermediates, (b) the demonstration of precursor-product relationships between putative intermediates, and (c) purification and characterization of enzymes involved in the metabolic interconversions. These criteria will be applied in our evaluation of the experimental evidence that supports the operation of a multibranched Chl b biosynthetic pathway in green(ing) plants.

    A. Determination of Precursor-Product Relationships In Vivo

    PPR2.gif - 7.39 K

    Fig. 7. The Three Possible Irreversible Precursor-Product Relationships Between Two Precursors (P, A) and One End Product (B) (Adapted from Rebeiz et al, 1988)


    In discussing the Chl b biosynthetic pathway, use will be made of kinetic analysis of precursor-product relationships in vivo. In 1988, equations were derived to investigate possible precursor-product relationships in branched, and interconnected pathways (Rebeiz et al, 1988, Tripathy and Rebeiz, 1988). It was shown that for any two compounds A and B, formed from a common precursor P such as ALA, and having a possible direct precursor-product relationship between them, for any number of time intervals t1 to t2, the following equation describes the relationship between the specific radioactivity of compound A, possible radiolabel incorporation from compound A into compound B, and the net synthesis of compound B from compound A (Rebeiz et al, 1988):

    QB2 = (*A1 + *A2)/2).(*B2) (Eq. 1)

    where:

    QB2 = amount of radiolabel incorporated into compound B during time interval t1 - t2;
    *A1,*A2 = specific radioactivity of compound A at the beginning and end of time interval t1 - t2 respectively.
    *B2 = amount of compound B synthesized during time interval t1 - t2.

    By comparing expected radiolabel incorporation into compound B, as calculated from Eq. 1, with experimentally determined incorporations into compound B, it is possible to tell whether compound B was formed exclusively from compound A or not. If compound B is formed exclusively from compound A, then within the range of experimental error, the theoretical and experimental radiolabel incorporations into compound B should be identical or reasonably similar. On the other hand, if compound B is not formed from compound A, or is partially formed from compound A, then the calculated and experimental radiolabel incorporations into compound B will be different. The difference between the calculated and experimental values, may then depend, among other things, on the extent of partial contribution of compound A to the synthesis of compound B.

    If comparison of calculated and experimental results indicates that compound B is not totally formed from compound A via pathway 1 (Fig. 5), then the question arises as to whether compound B is formed via pathway 2 or 3 (Fig. 5). Furthermore if compound B is found to be formed via pathway 2, then the contribution of compound A to the formation of compound B needs to be assessed. The determination of whether compound B is formed via pathway 2 or 3, can be achieved from conventional in vitro investigations of precursor product relationship between compound A and compound B. In other words, if pathway 2 is found to be operational then the contribution of compound A to the formation of compound B can be assessed from Eq. 2 (Rebeiz et al, 1988):

    %Conversion = 100 - [(|Exp-QBX|/Exp)100] Eq. 2.

    where:

    % Conversion = maximum possible percent conversion of compound A to compound B during any time interval X.
    Exp = actual 14C-incorporation into compound B by the end of time interval X, as determined experimentally.
    QBX = theoretical 14C-incorporation into compound B by the end of time interval X, as calculated from Eq. 1.
    |Exp-QBX| = absolute difference between the experimental and theoretical 14C-incorporation of precursor P into compound B during time interval X.

    B. Source of Oxygen During the Formation of the Formyl Group of Chl b

    Mass spectra of [7-hydroxymethyl]-chlorophyll b extracted from leaves greened in the presence of either 18O2 or H218O2 revealed that 18O was incorporated only from molecular oxygen into the 7-formyl group of Chl b (Porra et al, 1993; 1994). The high enrichment using 18O2, and the absence of labelling by H218O2, demonstrated that molecular oxygen is the sole precursor of the 7-formyl oxygen of chlorophyll b in greening maize leaves. This in turn suggested that a mono-oxygenase is involved in the oxidation of the methyl group to a formyl.

    E. References



    1. Rebeiz, C. A., J. M. Mayasich and B. C. Tripathy (1988). Chloroplast Biogenesis 61: Kinetic analysis of precursor-product relationships in complex biosynthetic pathways. J. Theor. Biol. 133: 319-326.
    2. Tripathy, B. C. and C. A. Rebeiz (1988). Chloroplast Biogenesis 60: Conversion of divinyl protochlorophyllide to monovinyl protochlorophyllide in green(ing) barley, a dark monovinyl light divinyl plant species. Plant Physiol. 87: 89-94.
    3. Porra, R. J., W. Schafer, E. Cmiel, I. Katheder, and H. Scheer (1993). Derivation of the formyl-group oxygen of chlorophyll b from molecular oxygen in greening leaves of higher plants (Zea mays). FEBS. 323: 31-34.
    4. Porra, R. J., W. Schafer, E. Cmiel, I. Katheder, and H. Scheer (1994). The derivation of the formyl-group oxygen of chlorophyll b in higher plants form molecular oxygen. Achievement of high enrichment of the 7-formyl-group oxygen form 18O2 in greening maize leaves.

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