All tetrapyrroles that will be discussed in this review, are derivatives either of Porphin or Phorbin . Because of its simplicity, the Fischer nomenclature and numbering systems will be used throughout this overview.
In higher plant thylakoids, the reactions between ALA and Proto are considered to take place in five different environments and may involve both spatial and chemical biosynthetic
heterogeneities (Fig. 1, and Fig. 2 ). This hypothesis is based on the detection of
resonance excitation energy transfer from Proto to various Chl–protein complexes in multiple thylakoid environments (
Table 1). Also, on the basis of resonance excitation energy transfer from Proto to Pchlide a, Averina and coworkers have proposed the existence of at least two types of Chl biosynthesis
centers which differ in their ability of form ALA (Averina et al, 1993, Averina, 1998).
A. Biosynthetic Heterogeneity of Delta-Aminolevulinic Acid (ALA)
Delta-aminolevulinic acid (5-aminolevulinic acid) is the building block of all tetrapyrroles in nature. It is synthesized via a different pathway in animal cells and lower plants than in green plants.
In animal cells, ALA is formed by condensation of glycine and succinyl-CoA (Gibson et al , 1958) . The reaction is catalyzed by ALA synthetase and takes place in the mitochondria. The biosynthesis of succinyl-CoA from succinic acid is catalyzed by succinyl-thiokinase in the presence of Mg ++ and ATP, and takes place also in the mitochondria. ALA is exported to the cytoplasm for further metabolism (Granick, 1963). In animal mitochondria ALA is mainly destined for the biosynthesis of Proto and heme (Rebeiz et al, 1996)Chen et al, 1981), Scenedesmus obliquus (Klein and Senger, 1978), a unicellular green alga, and in Euglena gracilus (Beale et al, 1981), a unicellular green flagellate alga, two pathways using, either glycine and succinyl-CoA, or incorporating the whole C-5-skeleton of glutamate into ALA (see below) are functional in the biosynthesis of ALA. In Scenedesmus both pathways appear to contribute to Chl formation in the light. In Euglena, it was proposed that ALA synthesis via glycine and succinyl-CoA is responsible for non-plastid tetrapyrrole biosynthesis (Beale et al, 1981). These results stress the need to investigate in more depth the molecular basis and biological significance of the ALA biosynthetic heterogeneity in a wider range of lower and higher plants.
In higher plants ALA is formed from glutamic acid (Beale and Castelfranco, 1974) via three reactions (Kannangara et al, 1984). In a first reaction, glutamate- tRNAGlu ligase catalyzes the ligation of glutamate to tRNA in the presence of ATP and Mg++. In a second reaction, the glutamyl-tRNA complex is converted into a linear glutamate semialdehyde (GSA) by NADPH:Glu-tRNA(oxido)reductase (also called glutamyl-tRNA dehydrogenase) (Kannangara et al, 1984) or into a cyclic GSA (hydoroxyaminotetrahydropyranone, HAT for short) (Jordan et al, 1989). Finally, GSA aminotransferase converts GSA to ALA in the presence of vitamin B6 and pyridoxal phosphate. These reactions take place in the stroma of the plastid.
The understanding of ALA biosynthetic heterogeneity in higher plants is still at a primitive stage although reports are surfacing in support of that notion. For example a reported differential
inhibition of ALA formation by gabaculine in black pine (Pinus nigra L.) during seed germination (Drazic and Bogdanovic, 2000) strongly suggests
that in black pine, ALA is formed at least via two different routes, one of which is inhibited by gabaculine. Also Averina and coworkers have proposed the existence of at least two types of
Chl biosynthesis centers which differ in their ability to form ALA (Averina et al, 1993; Averina, 1998). Recently,
multiple resonance excitation energy transfer sites from Proto to various Chl-protein complexes have been detected in higher green plants
(Table 1), which led to the extension of the Proto biosynthetic heterogeneity all the way to ALA formation.
Therefore, in ( Fig. 1 ), ALA biosynthesis is proposed to take place in five different thylakoid environments, via
routes 2, 8, 10, 11 and 12. It is uncertain at this stage whether or not similar reactions in the 5 biosynthetic routes between ALA and Proto are catalyzed by identical enzymes or not.
In other words it is still uncertain whether the spatial heterogeneity of ALA formation is accompanied by chemical biosynthetic heterogeneity or not.
B. Biosynthesis of Porphobilinogen (PBG)
PBG is the precursor of uroporphyrinogen III (Urogen III) which is the precursor of all intermediates of the heme and Chl biosynthetic pathways. It is formed from two molecules of ALA; in the process two molecules of water are liberatedw. The dimerization reaction is catalyzed by ALA dehydratase also known as PBG synthase (Gibson et al, 1955; Schmid and Shemin, 1955). The enzyme binding sites of the two ALA substrates have been designated the A and P sites. The A site gives rise to the acetic side chain, while the P site gives rise to the propionic side chain of of PBG. The first substrate binds to the P site where it forms a Schiff base with the enzyme. The second ALA molecule interacts with the A site (Jordan and Seehra, 1980) to form an enzyme-two substrate complex. The precise mechanism by which the 5-membered PBG ring is formed from the enzyme-two substrate complex is till uncertain.
In E. coli ALA Dehydretase contains two metal binding sites that have been designated a and b ( Spencer and Jordan, 1994). The a-site binds preferentially a Zn 2+ ion that is essential for catalytic activity. The b-site is exclusively a transition-metal-ion-binding site thought to be involved in protein conformation.
Beyond the possibility that in higher plants PBG may contribute to the formation of Proto in five different
environments (Table 1), no specific efforts have been made to document the nature and extent of PBG
biosynthetic heterogeneity in plants.
C. Biosynthesis of Uroporphyrinogen III (Urogen III)
Urogen III is the universal precursor of all metabolic tetrapyrroles (Neve and Labbe, 1956). It is the branching point where the biosynthesis of vitmin B12 diverges from that of heme and Chl. Its biosynthesis from PBG requires the cooperation of two enzymes, PBG deaminase (Bogorad, 1958) and Urogen III synthase also known as cosynthetase (Bogorad, 1958). In E. coli PBG deaminase is coded for by the gene hemC (Thomas and Jordan, 1986). The apoprotein consists of 353 amino acids with a molecular weight of 34245. The active site contains two constitutive PBG molecules (dipyrromethane cofactor) attached to the apoprotein by a cysteine residue (Cys-242) (Hart et al, 1987; Jordan and Warren, 1987). In a first step, one PBG molecule binds to the deaminase. A covalent bond is formed between the second constitutive PBG molecule and the first PBG substrate, and one molecule of ammonia is released. This first condensation leads to the formation of ring A of Urogen III. This step is repeated three more times and results in the formation of an open chain tetrapyrrole which is displaced from the enzyme by water to yield 1-hydroxymethylbilane (HMBL) (also called preuroporphyrinogen) (Battersby et al, 1979; Jordan and Seehra, 1979; Battersby et al, 1983; Battersby et al, 1982). Hydroxymethylbilane is unstable and in the absence of the cosynthetase cyclises at neutral pH to yield Urogen I. In the presence of the cosynthetase, hydroxymethylbilane is rapidly converted into Urogen III (Battersby et al, 1982b). This reaction involves inversion of ring D of HMBL and cyclization with the release of one water molecule. In E. coli the cosynthetase is coded for by the gene hemD (Jordan et al, 1988). The apoprotein consists of 246 amino acids with a molecular weight of 27766. HemC and hemD occur in tandem and overlap by one base pair. In animal cells, Urogen III is formed in the cytoplasm (Rebeiz et al, 1996). In plant cells, PGB deaminase and the cosynthetase are loosely bound to the plastid membranes (Lee et al, 1991).
Beyond the possibility that in higher plants Urogen III may contribute to the formation of Proto in five different
environments (Table 1), no specific efforts have been made to document the nature and extent of
Urogen III biosynthetic heterogeneity in plants.
D. Biosynthesis of Coproporphyrinogen III (Coprogen III)
Coprogen III is the precursor of protoporphyrinogen IX. It is formed from Urogen III by decarboxylation, a reaction catalyzed by Urogen decarboxylase which converts Urogen III to Coprogen III (Granick and Mauzerall, 1958; Mauzerall and Granick, 1958). Stepwise decarboxylation of the 4 acetate side chains and the resulting structures of the intermediates led to the proposal that the acetate side chains on rings D, A, B. and C are decarboxylated in a clockwise fashion starting with ring D ( Jackson et al, 1976 ; Jackson and Ferramola, 1980). Although this appears to be the case in patients suffering from porphyria cutanea tarda, a random rather than an ordered decarboxylation appears to prevail in normal individuals (Luo and Lim, 1993). These obsrvations led to the proposal that the substrate binding site has such a flexible architecture that at low Urogen concentrations, decarboxylation may be ordered, while at high substrate concentrations it may be random (Akhtar, 1984). The DNA coding for Urogen III decarboxylase in humans (Romeo et al, 1986) and rats (Romana et al, 1987) has been cloned and sequenced. The human enzyme consists of 367 amino acids with a molecular weight of 40,831. In animal cells Coprogen III is formed in the cytoplasm (Rebeiz, et al, 1996). In plants Urogen III decarboxylase appears to be loosely bound to the plastid membranes (Lee et al, 1991).
Beyond the possibility that in higher plants Coprogen III may contribute to the formation of Proto in five different
environments (Table 1), no specific efforts have been made to document the nature and extent of Coprogen III
biosynthetic heterogeneity in plants.
E. Biosynthesis of Protoporphyrinogen IX (Protogen IX)
Protogen IX is the precursor of protoporphyrin IX (Proto). Conversion of Coprogen III to Protogen IX involves oxidative decarboxylation of the two propionate side chains on rings A and B and their conversion to vinyl groups (Sano and Granick, 1961). The mammalian enzyme has an absolute requirement for oxygen, but requires no reducing agent. Recent studies indicate that the mammalian enzyme is a dimer of two 37000 subunits (Kohno et al, 1993). The observation of harderopoprphyrinogen ( one vinyl at position 2 and one propionate at position 4) accumulation (Sano and Granick, 1961) and its subsequent isolation (Kennedy et al, 1970) led to the proposal that the decarboxylation of ring A occurs before that of ring B. The precise mechanism of oxidative decarboxylation is still uncertain. In animal cells, cytoplasmic Coprogen III is transported to the mitochondria in an ATP-dependent process where it is converted to Protogen IX (Rebeiz, et al, 1996). In plant cells, Coproporphyrinogen oxidase appears to be loosely bound to the plastid membranes (Lee et al, 1991).
Beyond the possibility that in higher plants Protogen IX may contribute to the formation of Proto in five different environments (Table 1), no specific efforts have been made to document the nature and extent of Protogen IX biosynthetic heterogeneity in plants.
Proto is the immediate precursor of Mg-Proto which is the first committed intermediate of the Chl biosynthetic pathway. The role of Proto as an intermediate in the Chl biosynthetic pathways was based on the detection of Proto in X-ray Chlorella mutants inhibited in their capacity to form Chl (Granick, 1948). It was conjectured that since the mutants had lost the ability to form Chl but accumulated Proto, the latter was a logical precursor of Chl. The unambiguous role of Proto as a precursor of all Mg-porphyrins and phorbins including Chl was established (a) by conversion of exogenous Proto to Mg-Proto monomethyl ester (Mpe) by Rhodopseudomonas spheroides in the presence of ATP and Mg (Gorchein, 1972) and (b) by conversion of exogenous 14C- and unlabeled-Proto to Pchlide a [the immediate precursor of Chlide a] in organello (Mattheis and Rebeiz, 1977), 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, 1 971b), and capable of the net conversion of exogenous ALA to Mg-Protoporphyrins and Pchlide a (Rebeiz et al, 1975).Poulson and Polglase, 1975, Jacobs, and Jacobs, 1982). The oxidation involves the removal of 4 peripheral (meso) hydrogens and two inner hydrogens from the pyrrole nitrogens. In aerobic organisms, oxygen is the oxidant. Removal of the hydrogens appear to be stereospecific (Battersby, et al, 1976). The enzyme has been purified to apparent homogeneity from bovine liver (Siepker et al, 1987). It appears to be a monomer with a molecular weight of approximately 65,000. The bovine enzyme has a tightly bound FAD prosthetic group. The plant enzyme has been partially characterized (Jacobs and Jacobs, 1987). Protox was recently purified form spinach chloroplasts ( Watanabe et al, 2000). Its molecular weight was estimated at 60 kDa by SDS-PAGE.
In cucumber cotyledon and barley leaves chloroplasts, multiple Proto sites appear to be involved in resonance excitation energy transfer to the Chl a of LHCII, the major thylakoid LHC antenna, to LHCI-680, one of the inner LHC antennae of PSI, to Chl a of CP47 and CP29, two PSII antennae and to LHCI-73 an inner PSI antenna. This has led to the conclusion that the various Proto pools exist in different environments in the thylakoid membranes (Kopetz, 2000). This observation together with an apparent ALA biosynthetic heterogeneity ( Averina et al, 1993; Averina, 1998), has led to the proposal of five different Proto biosynthesis sub-locations in thylakoid membranes (Fig. 1). It is uncertain at this stage whether or not the 5 sub-locations contain identical enzymes for the conversion of ALA to Proto. In other words it is still uncertain whether the spatial heterogeneity of Proto formation is accompanied by biosynthetic heterogeneity or not.