Formation of the cyclopentanone ring (ring E) during the proposed conversion of Mpe to Pchlide a was suggested in 1950 to involve a beta-oxidation of a putative methyl Propionate side chain to a 3-keto derivative (Granick, 1950b). Detection of intermediates between Mpe and Pchlide a involving putative DV and MV metal-free acrylic, hydroxy and keto derivatives in ultraviolet Chlorella mutants, also led to the proposal that in plants, the formation of DV and MV Pchlide a involves a beta-oxidation sequence of the methyl propionate of DV and novel MV Mpe, at position 6 of the macrocycle (Ellsworth and Aronoff, 1969). The DV and MV keto methyl propionate species would then cyclize to yield DV and MV Pchlide a (Ellsworth and Aronoff, 1969). This work was met with skepticism and the putative intermediates were considered to be artifacts. This feeling was reinforced by the inability of the techniques, used by Ellsworth and Aronoff as well as of other analytical techniques prevailing at that time, to detect the proposed MV substrate (MV Mpe), the proposed intermediates (DV and MV acrylic, OH, and keto intermediates), and one of the end-products (DV Pchlide a) in normal, green, lower and higher plants.
A new phase in the systematic study of the cyclopentanone ring formation was ushered by the introduction of powerful cell-free systems capable of the massive net synthesis of Pchlide a from exogenous ALA and tetrapyrrole substrates (Rebeiz et al, 1975; Mattheis and Rebeiz, 1977a; 1977b; Daniell and Rebeiz, 192a; 1982b; Tripathy and Rebeiz, 1986) and the development of sensitive analytical fluorescence methodologies which allowed the demonstration of the DV and MV heterogeneity of the metabolic pools between Mg-Proto and Chl a (Rebeiz, et al, 1994). Using adapted techniques, the reactions between Mpe and Pchlide a have been reinvestigated by P. A. Castelfranco and collaborators (Wong and Castelfranco, 1985; Walker et al, 1988). In a series of experiments involving the conversion of added substrates to Pchlide a, it was shown that (a) both the beta-OH and beta-keto methyl propionate derivatives of Mpe could be converted in organello to Pchlide a, (b) that substrates with unesterified and esterified propionic acid residues at position 7 of the macrocycle were active substrates (c) that both, the DV and MV beta-OH and beta-keto methyl propionate derivatives also served as substrates, (d) that 2-ethyl,4-vinyl analogs were inactive, (e) that the 6-methyl acrylate derivative was also inactive, (e) that only one of the two 6-beta-hydroxy enantiomers was active, (f) that the MV 6-beta-keto derivative was 4 times more active than the DV analog, (g) and that DV and MV Mg-Proto were equally active.
On the basis of the above results, it has been suggested that in plants, the formation of the cyclopentanone ring involves conversion of Mpe to beta-OH and beta-keto methyl propionate derivatives, with stereospecificity at the level of the beta-keto derivative. It should be emphasized, however, that due to the lack of absolute specificity of the putative enzymes involved in cyclopentanone ring formation, elucidation of the actual sequence of events that result in the formation of the cyclopentanone ring of Pchlide a will have to await the unambiguous identification of all the putative DV and MV intermediates between DV and MV Mpe and DV and MV Pchlide a, as well as purification of the involved enzymes. At this stage, it appears that the reactions between Mpe and Pchlide a require molecular oxygen and iron (Spiller et al, 1982), and are inhibited by N-ethylmaleimide, dithiothreitol, and beta-mercaptoethanol (Wong and Castelfranco). It has also been reported that the conversion of Mpe to Pchlide a requires both the membrane and stromal fractions of the plastids (Walker et al, 1991). In our hands however, excellent cyclopentanone ring synthetase activity is observed with isolated plastid membranes without the need of a stromal factor. The latter appears to be involved however, in the regulation of the proportion of DV and MV Pchlide a formation (Kim et al, 1997).
The DV nature of the DV Pchlide a component of the heterogeneous Pchlide a pool of higher plants was determined by chemical derivatization coupled to analytical fluorescence spectroscopy at 77 K (Belanger and Rebeiz, 1980a). The DV nature of the DV Pchlide a component was also confirmed by 1H nuclear magnetic resonance (NMR) and fast atom bombardment (FAB) mass spectroscopy (Wu and Rebeiz, 1984).
The specific formation of the cyclopentanone ring in a DV-enriched sequence of reactions was demonstrated by conversion of exogenous DV Mpe to DV Pchlide a in isolated cucumber etiochloroplasts (Tripathy and Rebeiz, 1986). The biosynthesis of DV Pchlide a was accompanied however, by the formation of 20.7 % MV Pchlide a. These results indicate that the formation of DV and MV Pchlide a in organello are tightly coupled, and caution against assuming that conversion of DV substrates to Pchlide a in organello is likely to yield DV Pchlide a exclusively. The exact sequence, and mechanism of the reactions that convert DV Mpe to DV Pchlide a is not completely understood and will have to await he unambiguous identification of all putative DV intermediates between DV Mpe and DV Pchlide a, as well as purification of the involved enzymes, a mandatory condition for studying reaction mechanisms.
A precursor-product relationship between DV Pchlide a and DV Chlide a was established by demonstrating the photoconversion of DV Pchlide a to Chlide a in etiolated cucumber cotyledons induced to accumulate DV Pchlide a exclusively (Duggan and Rebeiz, 1982a).
The MV nature of the MV Pchlide a component of the heterogeneous Pchlide a pool of higher plants was determined by chemical derivatization coupled to analytical fluorescence spectroscopy at 77 K (Belanger and Rebeiz, 1980a). The MV nature of the heterogeneous Pchlide a pool was also confirmed by 1H nuclear magnetic resonance (NMR)s and fast atom bombardment (FAB) mass spectroscopy (Shedbalkar et al, 1991).
The specific formation of the cyclopentanone ring in an exclusive MV sequence of reactions was demonstrated by conversion of exogenous MV Mpe to MV Pchlide a in isolated cucumber and barley etiochloroplasts (Tripathy and Rebeiz, 1986). The biosynthesis of MV Pchlide a took place without formation of DV Pchlide a. The exact sequence, and mechanism of the reactions that convert MV Mpe to MV Pchlide a is not completely understood and will have to await the unambiguous identification of all the putative MV intermediates between MV Mpe and MV Pchlide a, as well as purification of the involved enzymes. MV Pchlide a can also be partially formed by reduction of the vinyl group of DV Pchlide a at position 4 of the macrocycle to ethyl. The reaction is catalyzed by [4-vinyl] Pchlide a reductase (4VPideR) (Tripathy and Rebeiz, 1988). It is very likely that this vinyl reductase is distinct from [4-vinyl] Mg-Proto reductase, which converts DV Mg-Proto to MV Mg-Proto (Kim and and Rebeiz, 1996). Indeed, 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 a separate [4-vinyl] reductase exists which acts prior to DV Pchlide a and DV Chlide a vinyl reduction, and which is responsible for the accumulation of MV Mg protoporphyrins in plants (Kim and Rebeiz, 1996). Also, 4VPR is probably different from [4-vinyl] Chlide a reductase (4VCR), which converts DV Chlide a to MV Chlide a (Parham and Rebeiz, 1992, 1995). This is evidenced by the observation that etiolated tissues containing an extremely active 4VCR, can be induced to accumulate massive amounts of DV Pchlide a in the absence of any MV Pchlide a formation. Yet when DV Chlide a is made available, it is converted to MV Chlide a by 4VCR, in less than one minute (Parham and Rebeiz, 1992). These results indicate that the proposal of only one [4-vinyl] reductase of wide substrate specificity, acting at all levels of DV to MV reduction is unfounded (Whyte and Griffiths, 1993).
A precursor-product relationship between MV Pchlide a and MV Chlide a was established by demonstrating the photoconversion of MV Pchlide a to MV Chlide a in etiolated cucumber cotyledons enriched in MV Pchlide a (Belanger and Rebeiz, 1980b).