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duplication involving the histidine region has been found to occur in 0.0 1p/, to 0.1 “/,; of S. typhimurium populations. Generally, these duplications are unstable, due to circularization and recombination between internally homologous chromosomal regions ; furthermore they are counterselected in rich media. However in recombination deficient strains (ret-), these duplications are stable. That gene duplication occurs is also shown by molecular hybridization between the specific DNA segments Arg F and Arg I, of E. coli K12 [6], which code for almost identical polypeptide chains carrying the ornithine transcarbamylase activity. Hopwood [7] and Zipkas and Riley [8] speculated on the possibility of the existence of gene duplication involving the whole genome of microorganisms (Fig. 1). In this case, genes functionally related should be found localized in a special orientation, one in respect to the other, on the circular chromosome. Zipkas and Riley used for their studies the genes of E. coli that could be grouped in the three following categories : a) isofunctional enzymes catalyzing the same reaction; b) enzymes belonging to the same metabolic pathway or catalyzing reactions involving identical or similar substrates; c) related cellular structures or functions. The genes clustered in operons were not taken into account if they did not possess a biochemical function similar to that found in other genes. 125 genes can be arranged into one or the other of these three categories. Within each group, individual genes were located on the chromosome at distances corresponding to 180” and 90” one from another. A total of 92 genes were involved in either about 90” or 180” relationship or both, representing 74% of the genes considered, a highly significant proportion. The result can be explained by two genome duplications, the duplicated genes having followed an independent evolution under the influence of mutation and selection imposed from the environment. Mizobuchi and Saito [9] using similar criteria and expanding the definition of functionally related genes to genes coding for enzymes with similar reaction mechanisms, even though the metabolic functions are not related (such as aspartate transcarbamylase and ornithine transcarbamylase) have postulated an additional genome duplication. They have then extended the study to 246 genes and found that 155 are separated on the circle by 45” or multiple thereof. Eighty two percent of the 38 genes coding for isofunctional enzymes obey to this spatial relationship. If the above hypotheses are correct, the

REVIEWS The evolution of biosynthetic pathways G. N. Cohen, I. Saint-Girons and P. Truffa-Bachi Evolutionary relationships exist between isofunctional enzymes involved in the ‘aspartic acidfamily’ biosynthetic pathway in Escherichia coli K12. Antigenic homologies have also been detected between some other enzymes of the same biosynthetic pathway which catalyze reactions possessing similar mechanisms. More than 30 years ago, Horowitz [l] proposed that the first organisms which appeared on our ,planet were heterotrophs thriving on substances found already made in the rich oceanic soup. These substances had been synthesized under the then prevailing geological and atmospheric circumstances. After a certain time, if a nutrient A came to run short, the only organisms which could survive were those able to synthetize the missing substance from a ‘precursor B’already present in the environment. The same process was repeated when B became limiting and so on. The selective pressure responsible for the synthesis of biosynthetic enzymes acts through the capacity of using precursor molecules when an essential metabolite is exhausted. According to Horowitz, the biosynthetic pathways as we know them have been progressively built starting backwards from the final metabolite of the pathway, to rejoin molecules of the great energy yielding paths such as glycolysis or the tricarboxylic acid cycle. Horowitz [2] rejuvenated his hypothesis twenty years later, making it more explicit due to several discoveries made in the meantime such as the bacterial operons, allosteric inhibition, and to the hypothesis formulated by Lewis [3] of gene duplication as an important factor of evolution. Gene tandem duplication can be the result of unequal crossing-over between two identical chromosomes. Lewis [3] and Ohno [4] consider gene duplication as a The authors are at the Unite de Biochimie Cellulaire dir Npartement de Biochimie et GPnPti~ Microbienne de l’lnstitut Pasteur, 75724 Paris, Cedex 15, France.

compulsory prerequisite to evolution of functions within the same organism. As a matter of fact, the existence of two or more copies of the same gene allows the accumulation of mutations in some copies and eventually the generation of new functions while maintaining the original function in one of the copies. The underlying idea in Horowitz’s hypothesis is that operons, that is juxtaposed genes coding for a series of proteins whose functions have the same linality, are the result of gene duplication followed by mutations endowing the corresponding proteins with distinct catalytic functions. According to this hypothesis, the allosteric regulation of the first enzyme of a given biosynthetic pathway is not the result of a selective pressure leading to the creation of the allosteric site, the latter being conserved since its origin, the regulatory substance being the product of the last enzyme. The allosteric site is thus a kind of ‘memory’ of the catalytic site of another enzyme of the pathway. This interpretation does not explain why the intermediate enzymes of the metabolic chain have apparently lost the capacity to recognize the allosteric effector. What is the evidence of gene duplication? A classical case of gene duplication was described in Drosophila as early as 1925 [5] in the Bar mutants. In bacteria the evidence is more recent: small duplication of the lac genes, of suppressor genes, of the genes for glycyl-transfer RNA synthetase have been described in Escherichia coli and an extended duplication, covering 22 per 100 of the Salmonella typhimurium genome has been recently reported. Another large

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Homologies between the isofunctional enzymes

A Fig. I. Genome duplication.

products of the mutated copies may have retained a more or less pronounced homology with the product of their ancestor. The obvious mean to test such an homology is to compare the amino acid sequence of the suspected relatives, or to challenge the DNA coding for ‘relative enzymes’ in hybridization tests. Information concerning the primary structure of enough proteins from the same organism is still scanty; we have seen that the hybridization of the Arg F and Arg I DNA has given an important information forgeneduplication (it is interesting to note that the two genes lie 45” apart on the circular chromosome, a beautiful example in favour ofgenome duplication !). In the absence of structural information, the antibody-antigen interactions represent a good approach to the study of possible ontogenic relationships. It is well known that extensive homology of antigens is necessary to produce a crossprecipitation, since three or more antigenic determinants are needed to form a lattice with the antibodies. However using molecules of antibodies bound to a solid matrix (immunoadsorbents), the presence of a single antigenic determinant is sufficient for the formation of the complex, then allowing the detection of homologies that are not likely to be found by immunoprecipitations. We have investigated, using the immunoadsorbent techniques, the possible evolutionary relationship of some of the enzymes of the ‘aspartic acid family’ (Fig. 2) in E. coli K12. The rationale for our choice is based on the following grounds: 1) existence of isofunctional enzymes : three aspartokinases have evolved in E. coli each with different regulation of its synthesis and activity. Moreover, two of these activities are associated, resulting from a gene fusion, in a single polypeptide chain with the homoserine dehydrogenase activity [lo] ; 2) some of the enzymes of this pathway have been obtained as pure proteins and antibodies raised against each of them; 3) the amino acid sequence of the aspartokinase I-homoserine dehydrogenase I is carried out in our laboratory; 4) all but one of the enzymes leading from aspartate to threonine are organized into an operon ; 5) the existence of enzymes catalyzing reactions possessing similar mechanisms.

Immunoadsorbents prepared with antiaspartokinase I-homoserine dehydrogenase I antibodies are capable of binding also aspartokinase II-homoserine dehydrogenase II and aspartokinase III [ll]. The extent of cross-reactivity calculated from the molar equivalent of each of the ‘heterologous’ antigens bound, compared to the amount of ‘homologous’, is very high. This suggests strongly that the three aspartokinases derive from a common ancestor, rather than being the products of convergent evolution. If we now look for the chromosomal localization of the three genes coding for the three aspartokinases, we find that Thr A (coding for aspartokinase I-homoserine dehydrogenase I) and Met LA4 (coding for the aspartokinase II-homoserine dehydrogenase II) are 45” apart, a result in agreement with the hypothesis

0

-

L-aspartate

of evolution by genome duplication. The third gene, coding for aspartokinase III, Lys C, is located at a position which is not predicted by the model. Homologies between enzymes catalyzing reactions with similar mechanisms

Homoserine kinase and the aspartokinase belong to the phosphotransferases. Immunoadsorbents specific to homoserine kinase or to aspartokinase I-homoserine dehydrogenase I recognize both enzymes, showing the existence of strong homologies between these two proteins [I 11.It has been possible to show that the homoserine kinaseis homologous to the aspartokinase, not to the homoserine dehydrogenase part of the bifunctional enzyme. Antibodies raised against homoserine kinase crossreact also with aspartokinase III. One is tempted to conclude that homoserine kinase and the three aspartokinases derive from an ancestor gene through gene tandem duplication and genome duplication.

~~~---II---~~--1--1)----/

I*’

s

5 Homoserine phosphate

Aspartylphosphate

0

I I L-threonine

T

meso -diaminopimelate

Reaction

Name of the enzymes

Name of the corresponding genes

1

Aspartokinase I

1

Aspartokinase I I

thr A met L -

1

Aspartokinase I I I

-lys c

2

Aspartic semialdehyde dehydrogenase

3

Homoserine dehydrogenase I

asd thr A -

3

Homoserine dehydrogenase I I

4

Homoserine kinase

5

Threonine synthetase

6

Threonine deaminase

7

Cystathionine -fl-synthase

8

Cystathionase

Fig. 2. Biosynthesis of the aspartate family.

met M thr B -thr C -ilv A -met B -met C

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Threonine synthase, the product of the ings of the 1975 Molecular Biology Meeting of reins, (Byron, V. and Vogel, H. J. eds.), pp. 15. Japan, pp. 60-62 Academic Press, New York and London third gene (Thr C) of the threonine operon 10 V&on, M., Falcoz-Kelly, F. and Cohen, G. N. Lewis, E. B. (1951) Cold Spring Harbor Symp. is not recognized by the antibodies raised (1972) Eur. J. Biochem. 28,520 Quart?. Biol. 16,159 against aspartokinase I-homoserine de11 Truffa-Bachi, P., Guiso, N., Cohen, G. N., Theze, Ohno, S. (1970) Evolution by Gene Duplication, hydrogenase I, or homoserine kinase. ConJ. and Burr, B. (1975) Proc. Nat. Acad. Sci. Springer-Verlag, Berlin U.S.A. 72, 1268 Sturtevant, A.H. (1925) Genetics 10, 117 versely, antibodies against threonine synKikuchi, A. and Gorini, L. (1975) Nurure 256, thase are unable to fix either aspartokinase 621 I-homoserine dehydrogenase I or homoHopwood, D.A. (1967) Bacterial. Rev. 31,373 serine kinase [ 121. Zipkas, D. and Riley, M. (1975) Proc. Nut. Acad. Homoserine kinase and aspartokinase I Sci. U.S.A. 72,1354 Mizobuchi, K. and Saito, H. (1975) in Proceedthat arise from a common ancestor gene are not adjacent on the chromosome : they are separated by the fragment of DNA coding for the carboxyl-terminal part of the bifunctional enzyme, that is, the homoserine dehydrogenase. This protein is the result of a gene fusion between two DNA fragments, each coding for the aspartokinase and the homoserine dehydrogenase respectively. In this respect, we are temptD. 0. Hall ed to speculate that this gene fusion took place before one of the genome duplicaThe process of photosynthesis is being re-examined and this well known example of a tions : this would explain in a simple manphotobiological energy conversion may have uses in providing fuel, food andfibre in the ner the existence of the other bifunctional enzyme, aspartokinase II-homoserine de- future. hydrogenase II. Light activates chlorophyll (photosyntheWe have also investigated other enPhotosynthesis sis), the retina (vision), DNA (mutations), zymes of the ‘aspartic acid family’; namephytochromes (day length control), the eye The unique capability that plant systems ly, we used antibodies against threonine and hypothalamus (orientation and navihave is to harvest light using their chlorosynthetase to investigate whether several gation in animals), the skin (tanning and phyll-containing membranes in order to enzymes of the pathway carrying out cancer) and a number of other similarly split water into its component parts, viz. carbon-oxygen or carbon-sulfur lyases important reactions in biology. But, in this oxygen and protons (hydrogen). Normally could have evolved from a common anarticle I want to try and outline the possithe plant uses the protons and ‘high cestor. bilities that photobiological energy conenergy’ electrons produced in the light Threonine deaminase is weakly or not version might have in providing fuel, food reactions to reduce CO, to the level of recognized by antibodies to threonine synand libre in the future - this is a recently carbohydrates (Fig. 1). This is a key reacthase. However, two of the enzymes of the revived concept of an old process, namely tion to life as we know it, additionally since methionine branch (Fig. 2) cystathionine photosynthesis. Since the change in oil the oxygen is a by-product of water split/I-synfhase (coded by Met B) and cystaprices three years ago there has been a ting. The carbon dioxide is fixed in the thionase strongly cross-react with antirenewed interest in solar energy systems as form of organic compounds as diverse as threonine synthase (Saint-Girons, I. et al., one of the renewable mechanisms for procarbohydrates, fatty acids and proteins in preparation). (and many other organic compounds In conclusion, the utilisation of a viding energy now and in the long term. which only plants can synthesize). I do not Solar energy also encompasses the idea of branched pathway is an obvious complicawish to go into the mechanism of phototion for an analysis of the evolution of using biological systems to capture the synthesis but one can consult numerous solar energy in a stored form. biosynthetic pathways. In addition, transbooks on the subject [12,13]. One of the important attributes of plants locations and gene fusions, stabilized by The theoretical efficiency of photois that they are able to collect diffuse solar the selective advantage conferred by posisynthesis in red light is 33% but crops radiation and store it for later use. We tion effects causing common regulation grown with good agriculture in temperate know that solar energy is ubiquitous and with other genes, bring further complicazones have efficiencies between 0.5 and 1% occurs universally to varying extents tions which render very difficult the proof throughout the world but the problem is (fixed carbon energy compared with total of Horowitz’s hypothesis in its original capturing it and storing it in a usable light energy available) and in tropical areas form. Nevertheless, using immunological form. Plants solved this problem via the between 0.5 and 2%. However, over the tools, we have shown that enzymes belongmechanism of photosynthesis when the whole earth the efficiency of photosyning to the same biosynthetic pathway thesis is only 0.1%. Even with this efficould derive from common ancestors: 1) blue-green algae developed the process about 3000-million years ago. It seems ciency the amount of carbon fixed every enzymes of the same operon ; 2) enzymes time that we reexamine how plants do it, year into stored energy is ten times the obeying different regulations but carrying try to improve plant efficiencies and even world’s use of energy in 1970 (Fig. 2). Of out identical reactions; 3) enzymes obeytry to emulate plant photosynthetic sys- this fixed energy only 0.5% ,is consumed ing different systems of regulation, but tems. (Consult references l-l 1 for general by our present world population. Thus one catalyzing similar reactions. reviews on solar energy and biological can see that vast amounts of energy are available in a fixed form and there is an approaches.) References excess of food available. The problem is D. 0. Hall ts Professor of Biology in the School of 1 Horowitz, N.H. (1945) Proc. Nat. Acud. Sci. that the distribution of this plant material Biological Sciences at King’s College, University of U.S.A. 31, 153 London, U.K. 2 Horowitz, N.H. (1965) Evolving Genes and Prois not generally where it is required in the

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