# The Pathways For Biosynthesis Of Tetrapyrrole

Олег Викторович Мосин

Tetrapyrroles are compounds whose molecules have four rings of the pyrrole type, generally linked together by single-atom bridges between the alpha positions of the five-membered pyrrole rings. Tetrapyrroles usually function as a metal-binding cofactor in many important enzymes, proteins and pigments, such as heme, chlorophyll, and cobalamine (vitamin B12). The tetrapyrrole biosynthetic pathway beginning with glycine and succinyl-CoA is found in animals, fungi, apicomplexan protozoa and bacteria. However, in some bacteria including Escherichia coli, tetrapyrrole biosynthesis begins with glutamate instead of glycine. This variation is also present in plants, who biosynthesize tetrapyrrole via glutamate for use as heme and chlorophyll.

THE PATHWAYS FOR BIOSYNTHESIS OF TETRAPYRROLE.

Oleg V. MOSIN

Moscow State Academy of Fine Chemical Technology, Moscow, Vernadskogo prospect, 86

Tetrapyrrole biosynthesis comprises as well the formation of non-cyclic, open chain tetrapyrroles like bile pigments in animal tissue and phycobilins and phytochrome in the plant kingdom, as the synthesis of cyclic tetrapyrroles like heme in all kind of living cells, chlorophylls in plants and corrins in certain microorganisms.

From the early studies on heme biosynthesis the intermediates of the heme pathway and the corresponding enzymes are well known. The pathway starts with the condensation of glycin and succinyl-CoA by ALA-synthase (Neuberger, 1961; Jordan and Shemin, 1972) to form ALA, the first specific intermediate of all tetrapyrroles. This pathway is called the Shemin-pathway. Subsequent condensation of two molecules of ALA leads to porphobilinogene, a step catalyzed by ALA-dehydratase (Shemin, 1972).

The formation of the first cyclic tetrapyrrole structure is then performed by two enzymes, porphobilinogen deaminase and uroporphyrinogen III cosynthase (Bogorad, 1958 a,b) leading to uroporphyrinogen III, one of the most common intermediates in the field of tetrapyrrole synthesis.

The next step is then the decarboxylation of uroporphyrinogen III to coproporphyrinogen III by uroporphyrinogen decarboxylase. Oxidation of coproporphyrinogen III by coproporphyrinogen oxidase leads to protoporphyrinogen IX, which is subsequently oxidized by protoporphyrinogen oxidase, under certain circumstances also nonenzymatically by oxygen. The last step before the branching into different heme-chromophores is the incorporation of Fe into the molecule by heme synthase, also called ferrochelatase.

The intermediates of heme biosynthesis and the enzymes catalyzing the interconversions are shown in Fig. 1. The branching of this pathway, leading to the formation of corrins and phycobilins will be discussed in detail in one of the following chapters.

As chlorophyll, the most spread biomolecule on earth, is also a cyclic tetrapyrroleand resembles hemes very much it was assumed for a long time that the pathway to chlorophyll was identical with the Shemin-pathway leading to hemes. But when attempts were made to demonstrate the presence of ALA-synthase in plants, the key-enzyme of the Shemin-pathway, could not be detected there.

This lead to the conclusion that another pathway for ALA-and thus chlorophyll-formation must be working in plants. The existence of this new pathway to ALA and chlorophyll was first proven by Beale and Castelfranco (1973, 1974a,b).

In these papers they clearly demonstrated by C-labelling experiments that the intact C-5 skeleton of glutamate or 2-oxoglutarate is incorporated into ALA without the loss or exchange of one of the C-atoms.

From this it was generally concluded that the C-5-pathway is restricted to plants whereas the classical Shemin-pathway only existed in animals and bacteria. This would mean that on one hand in plants the total amount of tetra-pyrroles derives from the ALA formed via the C-5-pathway and that on the other hand animals and bacteria cover their tetrapyrrole requirement via the Shemin-pathway.

This assumption was first ruled out by Klein and Senger (1978) who could demonstrate that in the pigment mutant C-2A of the green alga Scenedesmus obliquus both pathways are working. Under the influence of levulinic acid, a competitive inhibitor of ALA-dehydratase, great amounts of ALA are accumulated. The accumulated intermediates are both labelled by glutamate and 2-oxoglutarate via the C-5 pathway and by glycin and succinate via the Shemin pathway.

Both pathways are shown in Fig. 2 including the compartimentation of the reactions.

By specific 14C-labelling experiments with 1- and 5- C-glutamate and identically labelled 2-oxoglutarate and subsequent cleavage of the accumulated ALA it could be excluded that the label was incorporated via the transformation of 1 4 the C-5 compounds to succinate. In the case of 1- C-14 glutamate and 1-14C 2-oxoglutarate the specific radioactvity in ALA compared to labelling by 5- C-compounds should be very low and occur by random label only, when 14C-ALA derives from succinate. When ALA formed from C-1- precursors was cleaved by periodate the label was exclusively found in the C-5 of ALA which was the C-1 of the precursors. The presence of the Shemin-pathway in the same organism could also be reconfirmed by labelling experiments and in vitro enzyme assays.

The coexistence of the two pathways in one organism lead to reconsideration of the assumption that the pathways are restricted to only one kind of organism. By now it is generally accepted that both pathways possibly are present in animals, plants and facultative photosynthetic organisms (Klein and Porra, 1982). When chlorophyll formation was not inhibited by levulinic acid labelling of the chlorophylls via both pathways was found (Klein and Senger, 1978). Thus it was concluded that both pathways contribute to chlorophyll formation, the intensity of contribution being regulated by internal factors. When Oh-Hama et al. (1982) however cultured green algae with C-labelled precursors of both pathways, they could clearly demonstrate by C-NMR spectroscopy that only the C-5 pathway labelled at the expected positions in the porphyrin skeleton of chlorophyll, whereas glycin labelled chlorophyll only at the methyl group of the methylester adjacent to the iso-cyclic pentanone ring via the normal methyl metabolism.

This result obtained with green algae could be reconfirmed by Porra et al. (1982) for higher plants. As well as in algae in higher plants glycin labels only via 5-adenosyl-methionine at the methylester group attached to C-13.

Further evidence for the presence of both pathways was given by the observed labelling pattern (Riihl, 1984). By radioactive labelling with different precursors of both pathways the results of Oh-Hama et al. (1982) and Porra et al. (1983) could be reconfirmed and quantitatively reinsured by this different method. The effectiveness of labelling via the C-5 pathways could as well be demonstrated as the fact that there is no real label via the Shemin pathway into chlorophyll, but that this incorporation occurs via the methylmetabolism. In this case the presence of label in only the methylester position at C-13 was shown by reesterification with unlabelled methanol, resulting in a strong loss of radioactive label in the formed methylpheophorbide molecule.

A summary of these results is given in Tab. 1

lab.1 Labelling of ALA, phaeophytin a and methylphaeophor-bide a by different 14C-labelled precursors of both C-5- and Shemin-pathway.

Radioactive precursors Biosynthesis Organism

1-14C-glycin Unspecific Maize

2-14C-glycin Shemin Maize

1-14C-oxoglutarate C-5 Maize

1-14C-glycin Unspecific C-2A mutants

2-14C-glycin Shemin C-2A mutants

1-14C-oxoglutarate C-5 C-2A mutants


Further steps of chlorophyll biosynthesis from the first unequivocally identified precursor of chlorophyll formation, ALA, seem to be much more clear. Fig. 3 shows the pathway from ALA to chlorophyll with the principal intermediates of this pathway. It is taken from Castelfranco and Beale (1983).

After stepwise condensation of two molecules ALA to porphobilinogen (Jordan and Seehra, 1980) an instable hydroxymethylbilane (Battersby et al., 1979; Scott et al., 1980) is formed by the enzyme porphobilinogen deaminase. Inversion of ring D of the linear bilane and following closure of the tetrapyrrole cycle to yield uroporphyrinogen III is performed by uroporphyrinogen III cosynthase (Battersby et al., 1981). Decarboxylation of uroporphyrinogen III to coproporphyrinogen III by uroporphyrinogen decarboxylase (Jackson et al., 1976) is followed by oxi-dative decarboxylation of two propionic acid groups to vinyl groups by coproporphyrinogen III oxidase to form protoporphyrinogen IX (Games et al., 1976). Finally proto-porphyrin IX is formed by protoporphyrinogen oxidase by the removal of six electrons (Jacobs et al., 1982). The next steps lead to magnesium protoporphyrin IX monomethyl-ester and/or magnesium protoporphyrin IX, catalyzed by magnesium chelatase (Pardo et al., 198O; Richter and Rienits, 198O, 1982,and Fuesler et al., 1981).

The next steps, isocyclic ring formation, protochlorophyl-lide reduction and phytylation will not be discussed here in detail. For review see Castelfranco and Beale (1983).

As the formation of ALA via the Shemin-pathway is already elucidated and there is no doubt about further steps of tetrapyrrole biosynthesis via this pathway focus now lies on the early intermediates of the C-5 pathway which are still in discussion. In Fig. 4 (DSrnemann and Senger, 1980) all possible intermediates of the C-5-pathway are presented as earlier proposed by Beale et al. (1975) but today discussion focusses on only two of the possibilities: the route via glutamate-1-semialdehyde as proposed by Kannangara and Gough (1978) and the route via 4,5-dioxo-valerate (DOVA) as demonstrated by Dornemann and Senger (1980).

Kannangara and Gough (1978) could show that plastids isolated from barley can transfer glutamate to ALA. 2-Oxo-glutarate was converted at lower rate. From the ATP dependence of this conversion the authors concluded that via glutamate- 1-phosphate glutamate-1-semialdehyde should be formed and thus be the missing intermediate in the conversion of glutamate to ALA, although it must be stated that glutamate-1-semialdehyde could never be isolated from any organism.

The chemical synthesis of this compound, as described by Kannangara and Gough (1978) was found to be impossible by this and many other methods (Meisch et al., 1983; Kah, 1984).

Harel and co-workers (1983a,b) could also demonstrate the formation of ALA in isolated plastids from etiolated maize which was before isolation illuminated for 9O min, from glutamate, 2-oxo-glutarate and 4,5-dioxovalerate. From their experiments they conclude that DOVA is formed and may be an intermediate with the diversion of ALA to respiratory metabolism.

Evidence for the intermediate role of DOVA in the C-5-path-way was given by Dornemann and Senger (1978, 1980), who could demonstrate that under the competitive inhibition of ALA the accumulation of DOVA was paralleling the accumulation of ALA by a factor of 1/5 over a period of 24 hours in green algae. Radioactive label in DOVA was found to be 18 times higher via the C-5-pathway than by 1a reversible transamination of ALA formed from 2-14C-labelled glycin via the Shemin-pathway (Klein et al., 1978). Formerly DOVA-transaminase was discussed to be more or less reversible (Klein, 1978), but these experiments were performed with crude cell homogenates and might have included an unspecific transaminase reaction. Recent results however (Kah, 1984) showed that DOVA-transaminase, purified to homogeneity, was only working in the direction of ALA, reconfirming the former results of DOVA labelling.

In these experiments it was also be demonstrated by in vitro labelling with 1-14C-glutamate and 1-14C-2-Oxoglutarate that 5-14C-labelled DOVA and ALA were formed.

The ATP- and NADPH-dependence of the reactions was also demonstrated, NADH giving only 40% of NADPH response. Thus it has to be concluded that DOVA is the intermediate in the C-5-pathway in algae. Corresponding experiments in maize seem to point out in the same direction and will be reported soon as well.

Formerly branching of tetrapyrrole biosynthesis was understood without any problem because only one pool of ALA formed via the Shemin-pathway had to be reconsidered, followed by a chain of reactions to yield all types of tetrapyrroles. Under the viewpoint of two pathways to ALA and the assumption of their compartimentation problems of branching became more differentiated and questions of possible pool interchanges had to be discussed.

A general scheme of branching of tetrapyrrole biosynthesis is given in Fig. 5.

A first intercrossing of both pathways seems to be possible on the level of ALA. ALA-pools are able to interchange, but these conditions are more or less unphysio-logical or stress situations (Riihl, 1984). Thus the first branching point in the prolonged Shemin-pathway is urq-porphyrinogen III, the compound from which corrin biosynthesis starts (for review see Battersby and McDonald, 1982) .

This very important group of tetrapyrroles includes a great variety of very important compounds in regulation of cell metabolism like vitamin B^/ also known as cyano-cobalamine, methylcobalamin or adenosyl cobalamin, all these compounds being derivatives of cobyrinicacid, the key substance in cobalamin biosynthesis.

The next point of branching is reached at the level of protoporphyrin IX. From this compound in tetrapyrrole biosynthesis again a great variety of biologically important chromophores is derived: the group of phycobilins (for review see McDonagh, 1979 and Bennet and Siegelman, 1979) and phytochrome (for review see Kasemir, H., 1983).

Phycobilins, non cyclic tetrapyrroles, are accessory pigments in chlorophyll-b-less blue green- and red algae and form the "light-harvesting-complex" of the algae, the phycobilisomes. Phytochrome plays a great role in photomorphogenesis of higher plants, whereas in algae blue light effects seem to control this phenomenon. Some other lectures in this symposium will deal with these chromophores.

The C-5 pathway, in plants mainly leading to chlorophylls shows in methanogenic bacteria a very interesting branching to a nickel-containing tetrapyrrole. This chromophore was called FrfOQ and is directly involved in the formation of methane in these organisms. The structure of the chromophore was clarified by the groups of Thauer and Eschen-moser (1983).

That uroporphyrinogen is the point of branching could be shown by Gilles and Thauer (1983) by enzyme studies. It became also clear that the carrinoid pathway up to Sirohydrochlorin is followed and that afterwards the corphinoid structure of this nickel porphyrin is build up. Which early precursor to ALA is involved in this pathway was also clarified by Gilles et al. (1983) who could demonstrate that labelled succinyl-CoA via reductive carboxylation to 2-oxoglutarate is incorporated into ALA in this archae bacterium and is then further metabolized to uroporphyrinogen III. As glycine did not yield significant label in ALA and uroporphyrinogen III it has to be concluded that this new tetrapyrrole is formed via the C-5 pathway, demonstrating, that also in non photosynthetic bacteria the C-5 pathway is working.

For the first time Kannangara and Gough (1977)could show that isolated chloroplasts were able to form ALA and chlorophyll from glutamate, indicating that the C-5 pathway in higher plants is located in the chloroplast. These results from barley chloroplasts could be reconfirmed by Harel and Neeman (1983) with maize plastids and with green algae and maize chloroplasts by Riihl (1984).

As in cell-free extracts from both maize and Scenedesmus ALA-synthase activity could be measured (Riihl, 1984) the question of compartimentation of this ALA providing system arose. It could be shown that in isolated maize plastids no ALA-synthase activity was present. The assumption that this enzyme could in plants be located in the mitochondria could not be verified (Riihl, 1984). When mitochondria were isolated from both organisms even by radioactive assay no ALA-synthase activity could be detected in the mitochondria so that it seems to be reasonable that the Shemin-pathway is located in the cytoplasm of plant cells (Fig. 6). This is in contradiction to animal cells where ALA-synthase is described to be a mitochondrial enzyme (Granick and Sassa, 1971). The further enzymes up to Coproporphyrinogen III oxidase are located in the cytoplasm. The final steps up to heme are located again in the mitochondria.

Control and compartimentation are shown in Fig. 6, which is taken from Tait (1979).


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