Reprinted from New Beer

Transcription

Reprinted from New Beer in an Old Bottle: Eduard Buchner and the Growth of
Biochemical Knowledge, pp. 173–198, ed. A. Cornish-Bowden, Universitat de
València, Spain, 1997
ENZYME EVOLUTION AND THE
DEVELOPMENT OF METABOLIC
PATHWAYS
Juli Peretó, Renato Fani,
José Ignacio Leguina and Antonio Lazcano
1. INTRODUCTION: ENZYMES
AND THE ORIGIN OF LIFE
When Adolph von Bäyer learned that Eduard Buchner had been
awarded the 1907 Nobel Prize in Chemistry for his discovery of zymase, he remarked that “this will bring Buchner fame, even though he
has no chemical talent” (Bloch, 1994). And yet, although Eduard
Buchner’s first observations of cell-free fermentation had taken place
during a trial run to obtain immunologically active bacterial proteins
in his brother Hans’ laboratory, they would eventually become one of
the cornerstones of the new science of biochemistry. Although by
then the study of soluble enzymes was already a well-developed area
of research, they were considered as biologically much less important
than protoplasm itself, and many still adhered to T. H. Huxley’s
assertion that “protoplasm, simple or nucleated, is the formal basis of
all life” (Huxley, 1870).
The recognition that cell-free fermentation was due to enzymatic
activity rapidly challenged the views of protoplasmic biology, and led
to a new mechanistic enzyme-centred theory of life (Kohler, 1971,
1972). Although different and sometimes opposing ideas on the actual
nature of the protoplasm coexisted (Ling, 1984; Welch, 1995), the study
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P ERETÓ, F ANI, L EGUINA AND L AZCANO
of life’s defining properties was understood by many as the physicochemical characterization of protoplasm, and had thus been incorporated into colloid chemistry (Olby, 1970). Not surprisingly, discussions
on the emergence of life had centred upon the origin of protoplasm,
and terms like “primordial protoplasm” or “primordial protoplasmic
globules” were used by many influential researchers like Ernst
Haeckel, whose ideas on the nature of the evolutionary process had a
profound influence that extended itself into several 20th century
theories on the origins of the first living beings.
One of the first to incorporate enzymes into the discussions of the
origin of life was Leonard T. Troland (1914, 1917), who proposed that
the first living system had been a primordial self-replicating enzyme
whose catalytic activity influenced its immediate environment. This
hypothetical entity was assumed to be the outcome of a rare chance
event. Troland (1914) expressed it as follows:
Let us suppose that at a certain moment in earth-history, when
the ocean waters are yet warm, there suddenly appears at a
definite point in the oceanic body a small amount of a certain
catalyzer or enzyme [...] and when one of these enzymes first
appeared, bare of all body, in the aboriginal seas it followed as
a consequence of its characteristic regulative nature that the
phenomenon of life came too.
Troland’s hypothesis was relatively short-lived, but his idea of a
living molecule eventually gave rise to the primordial gene-based
theory of the origin of life developed by Muller (1926) and subsequently refined into his influential nucleic acid-first hypothesis
(Kamminga, 1986; Lazcano, 1995a). On the other hand, by the mid
1920s the apparent conflict between the protoplasmic theories of life
and the enzyme-based theory had been eroded. As Eduard Buchner
wrote (Buchner, 1899, in Kohler, 1972),
The concept of “living plasma” is quite indefinite; generally
one understands by it a mixture of various proteins which
carry out life functions. Among these are probably enzymes
as such or zymogens. There is thus no real difference
between the enzyme and the plasma hypothesis.
Thus, when Oparin (1924, 1938) suggested his metabolism-centred
hypothesis of an heterotrophic origin of life, what he had in mind as
protoplasm was a system in which all the physiological functions
were mediated by enzymes, and not the old view of cells as mere
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EVOLUTION OF METABOLIC PATHWAYS
physicochemical aggregates in which the vital functions took place.
It is somewhat ironic that although the understanding of the
evolution of metabolism was one of the driving forces behind Oparin’s
biochemical approach, the main focus of the research on the origin of
life has shifted away from to the origin of biosynthetic routes. Since it
is generally assumed that the first organisms were derived from the
preformed organic compounds available in the primitive environment (Oparin, 1924, 1938), the emergence of biosynthetic pathways
allowed primitive organisms to become increasingly less-dependent
on exogenous sources of amino acids, bases, and other compounds
which may have accumulated due to prebiotic processes. The purpose
of this chapter is to review and update previous discussions on the
development of metabolic pathways (cf. Lazcano et al., 1992), and to
underline some of the major unsolved questions which in our opinion
exist in this field.
2. THE EMERGENCE AND EARLY
EVOLUTION OF METABOLISM
Although considerable efforts have been made to understand the
emergence of the first living systems, we still do not know when and
how life originated. As it is sometimes possible to correlate major
evolutionary changes with environmental conditions, several
attempts have been made to infer the conditions in which life arose by
studying the oldest known organisms. The fruitfulness of this
approach may be found in the ideas of Oparin (1924, 1938). By assuming that useful hints could be inferred from metabolic pathways existing in contemporary cells, he made a thoroughly comparison of the
basic biochemical processes and concluded that the first beings were
anaerobic heterotrophs. This idea led to the hypothesis of chemical
evolution and, eventually, to the development of prebiotic chemistry
and other related origin-of-life research (Miller et al., 1997). Although
it may be reasonable to assume that the first living system was
already endowed with the same basic properties as those observed in
extant cells, today we know that such extrapolations into the distant
past merit considerable caution. We are still far from understanding
the characteristics of the first living systems, which may have lacked
even the most familiar features found in extant cells, such as proteinbased catalysts and phosphodiester-backbone genetic macromolecules
(Lazcano and Miller, 1996).
The lack of a fossil record strongly hinders our understanding of
175
biochemical evolution, but there is evidence that the basic biosynthetic
pathways were stablished in a short geological time. Isotopic
fractionation studies of geological samples from the Akilia formation
near Isua, Greenland, life may have existed 3.85 ! 109 years ago
(Mojzsis et al., 1996). The Apex sediments in the Australian Warrawoona formation show that highly diverse microbial communities
which may have included cyanobacteria were thriving on the
primitive Earth 3.5 ! 109 years ago (Schopf, 1993). As phylogenetic
trees based on small subunit rRNA suggest that cyanobacteria are a
relatively late branch of the eubacteria (Woese, 1987), early Precambrian life must have rapidly achieved levels of genetic organization,
biochemical complexity, and evolutionary potential comparable to
those of extant prokaryote populations.
Although it is not possible to assign a precise chronology to the
development of biochemical pathways, the possibility of their rapid
appearance is consistent with our current understanding of microbial
population genetics, which shows that the acquisition of new metabolic traits does not necessarily follows a slow evolutionary pace. Such
mode of evolution is consistent not only with the well-documented
rapid emergence and spread of antibiotic resistance factors, but also
with the experimental selection of variants that develop in few days or
weeks the ability to use novel sources of carbon and nitrogen (Lerner
et al., 1964; Clarke, 1974, 1983; Chapman and Ragan, 1980; Dijkhuizen, 1993; Hall and Hauer, 1993). Such directed-evolution experiments, which may also provide insights on the development of anabolic pathways, have shown that adaptation to novel substrates
typically involves deregulation and amplification of genes encoding
enzymes lacking absolute substrate specificity (Hall and Hauer, 1993).
These observations are consistent with the assumed properties of unregulated, primitive catalytic proteins with broad substrate specificity
that may have existed during early stages of evolution.
Protein sequence analysis and molecular have enhanced our
understanding of early cellular evolution, but their applicability
cannot be extended beyond a threshold that corresponds to a period in
which ribosome-mediated protein biosynthesis was already in operation. Older stages are not yet amenable to this type of analysis, but
important insights on the early trends in metabolic evolution can be
achieved by analysing the phylogenetic distribution of pathways over
a broad cross-section of biodiversity. Such backtrack characterizations
are hindered not only by polyphyletic secondary losses, lateral transfers, replacements, and redundancies of enzymatic steps and even of
entire metabolic routes that may have taken place, but also by our
176
choice of model organisms (Becerra et al., 1997). It is also possible that
enzymes may have participated in alternative pathways that no
longer exist or remain to be discovered (Zubay, 1993). Nevertheless,
comparative genomic analysis strongly suggest that the last common
ancestor, i.e. the cenancestor, of the three primary domains (Bacteria,
Archaea, and Eucarya) had already evolved the universal biochemical functions of primary metabolism, and was a rather sophisticated
cell probably much like modern prokaryotes. Thus, it must be considered the last of a long line of simpler earlier cells for which no
modern equivalent is known (Lazcano, 1995b).
The complete sequencing of an increasing number of complete
genomes is providing not only with lists of genes that can be used to
understand the differences and similarities among the Bacteria,
Archaea, and Eucarya, but also for consensus sequence reconstructions and inferences about cellular functions, which in some cases
may be linked to information about the early terrestrial environment.
However, it should be kept in mind that the functions of many open
reading frames derived from these sequencing projects are tentatively
identified only by computer searches based on structural similarity to
known sequences in databases. Furthermore, cladistic analysis of biosynthetic genes provides information on the phylogenetic history and
the properties of isolated components, but not necessarily on the
functional coordination between the components of multigenic traits
such as metabolic pathways.
3. DO BIOSYNTHETIC ROUTES EVOLVE BACKWARDS?
The first attempt to explain in detail the origin of metabolic pathways
was developed by Horowitz (1945), who, based on Oparin’s (1938)
heterotrophic hypothesis and on the one-to-one correspondence between genes and enzymes as suggested by Beadle and Tatum (1941),
suggested that biosynthetic enzymes had been acquired via gene
duplications that took place in reverse order as found in extant pathways. This idea, also known as the retrograde hypothesis, states that if
the contemporary biosynthesis of compound A requires the sequential
transformations of precursors D, C, and B via the corresponding
enzymes as in the following scheme,
c C!
b B!
a A
D!
then the final product A of a given metabolic route was the first com177
pound used by the primordial heterotrophs. When A became depleted
from the primitive soup, the transformation of a chemically related
compound B into A catalysed by enzyme a would lead into a simple,
one-step pathway. The selection of variants having a mutant b enzyme related to a via a duplication event and capable of mediating the
transformation of a molecule C chemically related into B, would lead
into an increasingly complex route, a process that would continue
until the entire pathway was established in a backward fashion
(Horowitz, 1945). Twenty years later, the discovery of operons prompted Horowitz (1965) to restate his model, arguing that it was supported
not only by the overlap between product and substrate of enzymes
catalysing succesive reactions, but also by the clustering of genes, that
could be explained by a series of tandem duplications.
The retrograde hypothesis establishes a clear evolutionary connection between prebiotic chemistry and the development of metabolic pathways, and may be invoked to explain some routes. For instance, the non-enzymatic formation under possible prebiotic conditions of threonine, methionine, and pyrimidines mimic to certain
extent the corresponding biosynthetic pathways (Ferris and Joshi,
1979; Yamagata et al., 1990; Keefe et al., 1995). If this model explains
orotate biosynthesis, then the instability of carbamoyl phosphate
under aqueous conditions would imply that the critical step in the
development of this route was the emergence of a primitive carbamoyl phosphate synthetase (Fig. 1).
However, the origin of many other anabolic routes cannot be
understood in terms of their backwards development as they involve
many unstable intermediates. It has been argued that the Horowitz
hypothesis also fails to account for the origin of catabolic pathway
regulatory mechanisms, and for the development of biosynthetic
routes involving dissimilar reactions (Hegeman and Rosenberg,
1970). Additional criticisms have been summarized elsewhere and
include the following (Lazcano et al., 1992):
1.
The Horowitz hypothesis is based on the assumption that all the
biochemical intermediates of the basic biosynthetic routes were
available in the prebiotic soup. However, most metabolic intermediates are chemically unstable (Cánovas et al., 1967; Ornston,
1971; Jensen, 1976), and their synthesis and accumulation in the
prebiotic environment appears unlikely.
2.
Many metabolic intermediates are anionic, phosphorylated compounds that could not permeate primordial membranes in the
178
Fig. 1. UMP biosynthetic
pathway. Compounds in boxes
are likely components of the
prebiotic environment. The
UV-induced decarboxylation of
orotic acid into uracil is
analogous to the enzymatic
decarboxylation of orotidylate
into UMP shown here (Ferris
and Joshi, 1979).
absence of specialized transport systems, whose presence cannot
be assumed in early cells (Ornston, 1971; Jensen, 1976).
3.
According to the retrograde hypothesis, successive steps in metabolic pathways would involve similar chemical reactions, but
frequently this is not the case (Wu et al., 1968; Clarke, 1974). Such
a criticism can be overcome if similar reaction mechanisms are
involved, even if the chemical changes are themselves of
179
different nature (Jeffcoat and Dagley, 1973; Clarke, 1974).
4.
If the enzymes catalysing successive steps in a given metabolic
pathway resulted from a series of gene duplication events
(Horowitz, 1965), then they must share structural similarities
(Hegeman and Rosenberg, 1970). The list of known examples
confirmed by sequence comparisons that satisfy this conditions
is small, and includes (a) the methionine biosynthetic enzymes !-cystathionase and cystathione !-synthase (Belfaiza et
al., 1986); (b) protochlorophyllide reductase and chlorin reductase, which are involved in bacteriochlorophyll biosynthesis
(Burke et al., 1993); and (c) the products of the hisA and hisF
genes, which participate in histidine anabolism (Fani et al.,
1995). A possible additional example may include the set
formed by N-phosphoribosylanthranilate isomerase, indoleglycerol-3-phosphate synthase, and the " subunit of tryptophan
synthase, which catalyse the three sequential steps between
phosphoribosyl anthranilate and indole in tryptophan biosynthesis. Three-dimensional structural comparisons of these proteins have shown that they all share an overall eightfold !!"
barrel motif, and that significant portions of their active sites are
superimposable, suggesting a common ancestry (Wilmanns et
al., 1991).
There is a possible variant of the retrograde hypothesis. The availability of decomposition products of organic compounds of abiotic
origin raises the possibility of the development of a biosynthetic pathway in a stepwise fashion, using the sequence of molecules available
in the decomposition route, rather than its precursors in the contemporary reaction sequence (Keefe et al., 1995). This is comparable to the
proposal by Degani and Halmann (1967), who argued that the
presence of fructose 6-phosphate, glyceraldehyde 3-phosphate, dihydroxyacetone and lactic acid as by-products of the non-enzymatic
alkaline degradation of glucose 6-phosphate is evidence of ancient,
pre-enzymic metabolic pathways. However, sugars and their phosphorylated derivatives decompose very rapidly on a geological timescale (Larralde et al., 1995), and glucose 6-phosphate is an unlikely
prebiotic compound.
This alternative interpretation of the Horowitz hypothesis has
been used in an attempt to explain the origin of branched-chain
amino acid biosyntheses (Keefe et al., 1995). Since their biological
precursors include !-keto acids, which readily undergo irreversible
180
decarboxylations and have short half-lives, a retrograde mechanism is
unlikely. However, deamination products of valine, isoleucine, and
leucine are isobutyric, !-methylbutyric and isovaleric acids respectively. The prebiotic availalibity of these short chain aliphatic acids is
indicated by their presence in the 4.6 ! 109 years old Murchison
meteorite (Lawless and Yuen, 1979). As summarized in Fig. 2, a
simple model explaining the synthesis of branched-chain amino
acids via a reductive carboxylation followed by non-enzymatic transamination can be easily derived, and may be tested experimentally
(Keefe et al., 1995).
Fig. 2. A model of semi-enzymatic synthesis of leucine from isovaleric acid. The
reductive carboxylation step could have been carried out by pyrite, low potential
ferredoxins, or other reducing agents (based on Keefe et al., 1995).
4. DO METABOLIC PATHWAYS EVOLVE FORWARDS?
An alternative forward model of metabolic evolution was suggested by
Granick (1957, 1965). Based on the Haeckelian belief that ontogeny
recapitulates phylogeny, he concluded that the biosynthesis of some
relatively complex end-products could be explained by forward evolution from relatively simple precursors. This model assumes that
simpler biochemical compounds are ancestral to more complicated
end-products, i.e. that the enzymes that catalyse earlier steps in a
given pathway are older than the latter ones. As the synthesis of
chlorophyll requires one fewer enzymatic reduction from chlorin
than does the formation of bacteriochlorophyll, Granick concluded
that bacteriochlorophyll anabolism had appeared in earlier photosynthetic organisms already endowed with chlorophyll. However, the
phylogenetic distribution of chlorophyll and bacteriochlorophyll does
not fit with Granick’s hypothesis. Sequence analysis of the iron-containing reductases involved in the biosynthesis of the two photopigments suggest instead that an ancestral bifunctional enzyme that
may have catalysed the two sucessive reductions on one substrate,
thus providing the ancestor of all photosynthetic eubacteria with a
bacteriochlorophyll-like photopigment (Burke et al., 1993).
As according to the Granick hypothesis the structure of a given
181
pathway reflects its history, it follows not only that the enzymes that
catalyse earlier steps in a given pathway are more ancient, but also
that each step of biosynthetic routes was selected because its product
had a function more useful that the precursor itself. The idea that
extant biochemical systems and metabolism recapitulate biogenesis
and can be used in backward extrapolations to the origin of life itself
have been presented by de Duve (1991) and Morowitz (1992) and will
not be discussed here.
The structure of several biosynthetic routes does suggest that their
development into successively longer pathways involved the progressive recruitment of enzymatic steps. Two obvious cases are the ATPdependent amination interconversion reactions that form asparagine
and glutamine. A more significant example may be deoxyribonucleotide biosynthesis. As underlined by Ferris and Usher (1983),
the retrograde hypothesis implies that DNA preceded RNA as cellular
genetic material. However, there is circumstancial evidence suggesting that DNA appeared in RNA/protein organisms (Lazcano et al.,
1988). What the structure of deoxyribonucleotide anabolism does
suggest is that their ribonucleotide reductase-mediated biosynthesis
may the outcome of the recruitment of an enzymatic step that was
added to a pre-existing biosynthetic pathway, i.e. that at least a portion
of this route may reflect its evolutionary development (Fig. 3).
The Granick hypothesis can also be invoked to explain the
anabolism of several membrane-components. One such case corresponds to the presence of the unsaturated ether core lipid 2,3-di-Ogeranylgeranyl-sn-glycerol, the major lipidic component of the
membrane of the ancient hyperthermophilic archaeon Methanopyrus
kandleri (Hafenbradl et al., 1993), and an anabolic intermediate in the
biosynthesis of 2,3-di-O-phytanyl-sn-glycerol, which is common in
other archaeal membranes. This reaction sequence may be interpreted as reflecting the progressively more complex route that evolved
by the terminal addition of enzymatic steps to an older pathway that at
one point ended in the synthesis of the unsaturated ether core lipid.
As phylogenies based on 16S rRNA place Methanopyrus kandleri on an
older branch than other archaea it can be argued that the structure of
the pathway mirrors the phylogeny of the group, i.e. that more
recently evolved organisms exhibit an more complex ether lipid
biosynthesis pathway (Stetter, 1996).
The oxygen-dependent biosynthesis of cholesterol suggests that
this forward mode of metabolic development was not restricted to the
182
Fig. 3. De novo biosynthesis of nucleotide
(based on Kornberg,
1980).
early stages of biological evolution. Cyclization of squalene into the
membrane-reinforcing cholesterol may have also evolved in a temporal discrete-step sequence driven mainly by adaptation to an
increasingly higher atmospheric oxygen concentration that took
place during Proterozoic times, 2.5 to 0.6 ! 109 years ago (Bloch, 1985;
Ourisson and Nakatani, 1994). The hypothesis that the first steps in the
biosynthesis of membrane-reinforcing compounds are the oldest ones
is consistent with the observation that cycloartenol, one of its
intermediates (Fig. 4), is a functional molecule that can replace
cholesterol both in vivo and in vitro. Thus, at a given point in time this
intermediate may have been the endproduct of the biosynthetic
pathway (Ourisson and Nakatani, 1994). A hypothetical sequence of
the progressive step-by-step evolution of cholesterol anabolism based
on the oxygen requirements of model organisms has been suggested,
which is also consistent with the enhanced stabilization of liposomes
observed when the progressively demethylated intermediates of the
cholesterol biosynthesis are added to them (Bloch, 1994).
183
5. THE PATCHWORK ASSEMBLY
OF BIOSYNTHETIC ROUTES
The significance of gene duplication in the development of biosynthetic innovations was discussed by Lewis (1951), who argued that
duplication followed by divergence underlines the origin of related
enzymes that take part in different metabolic routes. This idea was
developed by Waley (1969), who suggested that the stepwise evolution
of metabolic pathways was due to the accumulation of random,
Fig. 4. Cyclization of squalene-derived-2,3-oxide to cholesterol. The biosynthesis of
stigmasterol from cycloartenol is not shown.
184
minute genetic changes in duplicated genes, which would eventually
lead to conformational changes and the development of new enzymes
with similar biochemical properties. Similar hypotheses were
developed independently by Ycv as (1974) and Jensen (1976), who
argued that biosynthetic pathways may have been assembled by the
recruitment of primitive enzymes that could react with a wide range
of chemically related substrates. Such relatively slow, unspecific
enzymes may have represented a mechanism by which primitive
cells with small genomes could overcome their limited coding abilities (Ycv as, 1974). Such “patchwork” assembly of metabolic pathways
by the serial assemblage of inefficient catalysts can be considered as
an example of tinkering (Jacob, 1977), and is consistent with the
notion of a less accurate, error-prone primordial primitive translation
apparatus synthesizing small “statistical” enzymes (Woese, 1965).
According to this hypothesis, new enzymes result from amplification events followed by divergence, which lead to the narrowing of
their specificities, i.e. early enzymes may have been ambiguous catalysts using similar substrates and producing a family of related compounds. Evolution of duplicates during early stages may have been
influenced by the lack of DNA repair mechanisms combined with
high levels of ionizing and ultraviolet radiation in the early Precambrian environment (Koch, 1972). The gene recruitment model explains the diversification of ancestral enzymes of broad specificity into
families of related catalysts on the basis of point mutations, small
deletions, and other minute changes. Since prokaryotes are haploid
organisms, most of these small genetic changes would be expressed as
soon as they arose, resulting in a rapid mode of metabolic evolution.
However, the products of duplication events could be kept by primitive cells if gene conversion was avoided and new separate and
essential cellular roles appeared. It is not know how many modifications are needed for the appearance of new catalytic properties, but
directed mutagenesis studies suggest that at least in some cases few
genetic changes are actually required. For instance, site-specific substitution experiments have shown that replacement of one amino acid
can modify the substrate specificity of an eubacterial NAD-dependent
lactate dehydrogenase into a highly specific, active malate dehydrogenase (Wilks et al., 1988).
The recruitment of enzymes from different metabolic pathways
to serve novel catabolic routes is well documented under laboratory
conditions. These are the so-called “directed evolution-experiments”,
in which microbial populations are subjected to a strong selective
185
pressure and select phenotypes capable of metabolizing new substrates
(Clarke, 1983; Mortlock, 1992). The acquisition of new catabolic activities typically results from the loss of existing repressive control by
single mutations in regulator genes and the subsequent recruitment of
duplicates (Wu et al., 1978; Clarke, 1974; Mortlock and Gallo, 1992).
As reviewed by Jensen (1976), the patchwork theory is supported
by sequence comparisons showing that the two different carbamoylphosphate transferases involved in arginine and pyrimidine biosyntheses have evolved from a common ancestor. Additional evidence for
this mode of evolution has been discussed elsewhere (Lazcano et al.,
1992), and includes (a) the common ancestry of the Escherichia coli
flavin-dependent pyruvate oxidase involved in the decarboxylation of
pyruvate to acetate, with the acetohydroxy acid synthase that
participates in branched-chain amino acid biosynthesis (Chang,
1992); and (b) the experimental evidence that few mutations can alter
the preference of human gluthatione reductase for NADPH as cofactor. The results of structural studies that have demonstrated its homology with the E. coli thioredoxin reductase 3 and other proteins of the
same family, suggest that major specificity changes and reconfigurations of their catalytic sites are the outcome of small, simple alterations
of the relative spatial orientations of their different domains from a
common ancestor (Petsko, 1991; Schiering et al., 1991).
Additional examples of enzyme recruitment include (a) glycolysis (Fothergill-Gilmore and Michels, 1993), (b) nucleotide salvage
pathways (Becerra and Lazcano, 1997), and (c) histidine biosynthesis
(see below), The list is probably longer, as the existence of succesive
biochemical reactions catalysed by homologous enzymes does not
prove by itself neither the retrograde or the Granick hypotheses, since
during the early evolution of a given pathway different steps could
have been mediated by a primitive, less specific enzyme. Accordingly, the homologies discussed in the previous sections between (a)
protochloride and chlorin reductases (Burke et al., 1993), and (b) Nphosphoribosylanthranilate isomerase, indole-glycerol-3-phosphate
synthase and the ! subunit of tryptophan synthase (Wilmanns et al.,
1991), are also consistent with the gene recruitment model.
The repeated occurrences of homologous enzymes in different
pathways also provide evidence of patchwork tinkering. Data derived
from the ongoing genome projects with model organisms from the
three domains (Bacteria, Archaea, and Eucarya), has demonstrated
that a large portion of each organism’s genes are related to each other,
as well as to genes in distantly related species. All known life forms
share a common pool of highly genetic information that was shaped
186
to a considerable extent by gene duplications that took place prior to the
divergence of the three major cell domains (Koonin et al., 1995;
Labedan and Riley, 1995), and which includes many conserved and
easily identifiable biosynthetic genes.
As the patchwork hypothesis does not predicts the observed genetic linkage of enzymes in the same biosynthetic pathway (Ornston,
1971), a period during which major translocational events took place
must be assumed. This is consistent with comparative genomic
analysis that suggest the relative lability of prokaryotic gene order
(Mushegian and Koonin, 1996; St. Jean and Charlebois, 1996). The
universality of the patchwork mechanism is contradicted by biochemical redundancies of analogous character, such as the lack of
homology between (a) tryptophanase and the ! subunit of trytophan
synthase, two pyridoxal 5´-dependent enzymes that catalyse ! elimination reactions (Jensen and Gu, 1996); and (b) the F- and G-type
glutamine-amido transferases, which may not share a common
ancestor (Zalkin, 1985).
6. THE ORIGIN AND EARLY EVOLUTION
OF HISTIDINE BIOSYNTHESIS: A CASE STUDY
Histidine biosynthesis is one of the best studied anabolic pathways. A
large body of genetic and biochemical information is available,
including operon structure, gene expression, and increasingly large
sequence databases (Alifano et al., 1996). The apparently universal
phylogenetic distribution of his genes suggest that histidine
biosynthesis was already part of the metabolic abilities of the
cenancestor (Lazcano et al., 1992; Fani et al., 1995). Moreover, it is
generally accepted that histidine ("-amino-4-imidazole propionitrile)
plays an essential role in biological systems due to its imidazole
moiety, which underlies its function as a general acid-base catalyst by
proton relay mechanisms in a number of biochemical reactions.
The wide variety of imidazole-containing compounds that can be
formed non-enzymatically suggest that histidine and its possible
prebiotic analogues such as #-(4´-imidazolyl)-!-amino butyric acid
may have existed in the prebiotic environment. Following the synthesis of imidazole from a mixture of glyoxal, ammonia, and formaldehyde (Oró et al., 1984), it was shown that imidazole-4-glycol and
imidazole-4-acetaldehyde could be synthesized from erythrose and
formamidine (Shen et al., 1987). Additional experiments demon187
strated that imidazole-4-acetaldehyde could be directly converted to
histidine by a Strecker cyanohydrin synthesis (Fig. 5) involving the
hydrolysis of a hystidyl-nitrile intermediate (Shen et al., 1990c).
Synthesis of the dipeptide histidyl-histidine under possible primitive
conditions, involving the evaporation of an aqueous solution of histidine in the presence of condensing agents, has been achieved and is
an efficient process (Shen et al., 1990b). Histidyl-histidine is known to
catalyse the formation of peptide bonds and to promote the
oligomerization of glycine (White and Erickson, 1980), and of 2´,3´cyclic AMP under cyclic wet-and-dry laboratory reactions simulating
a primitive evaporating pond (Shen et al., 1990a). This body of data
supports the hypothesis that simple peptides of prebiotic origin
containing at least two imidazole groups may have acted as primitive
acid-base catalysts.
If histidine was required by primitive biological catalysts, then
the eventual exhaustion of its prebiotic supply must have imposed an
important selection pressure favouring those organisms capable of
synthesizing imidazole-containing compounds. Since histidine biosynthesis requires one carbon and one nitrogen equivalent from the
purine ring of ATP, it has been proposed that it may be the molecular
descendant of a catalytic ribonucleotide dating from an earlier bio-
Fig. 5. Strecker synthesis of histidine (based on Shen et al., 1990c).
188
chemical stage in which RNA played a central role in biological
catalysis (White, 1976). However, this proposal does not explains how
the histidine route was assembled, nor the origin of its enzymes (Alifano et al., 1996). The possibility that histidine biosynthesis evolved in
a retrograde fashion from intermediates present in the primitive broth
(Horowitz, 1965) is also unlikely, as (a) it is difficult to envision the
prebiotic synthesis and accumulation of the ribotide-derivatives involved in the metabolic route; and (b) there are no similarities
between histidine anabolism and its Strecker synthesis (Fig. 5). Thus,
at present it seems likely that a testable description of the emergence
of this pathway should invoke spontaneous chemical reactions leading to the formation of the imidazole ring and its condensation with
the alkyl-residue, as well as a coherent description of how primitive,
less specific biological catalysts may have taken over the entire
sequence of reactions (Alifano et al., 1996).
Cladistic analysis of the his genes has demonstrated the central
role played by paralogous duplications in its assemblage. The possibility that histidine biosynthesis was originally mediated by less specific enzymes has been reviewed elsewhere (Fani et al., 1995), and is
supported by sequence analysis which have demonstrated the homology of (a) ATP phosphoribosyltransferase (hisG) with all other phosphoribosyl transferases and with nucleosidases; (b) of imidazole
phosphate aminotransferase (hisC) with other pyridoxal-dependent
aminotransferases (Jensen and Gu, 1996); and (c) of imidazole glycerol phosphate (hisH) with all other G-type glutamine amidotransferases.
One of the most interesting aspects of histidine biosynthesis
revealed by sequence analysis is the common ancestry of the hisA
and hisF genes, which have resulted from two successive duplications involving first an ancestral module half the size of the extant
genes (Fani et al., 1994). The primitive pathway may have thus possessed an ancestral enzyme that catalysed two reactions on one substrate, assisting first in the isomerization of N´-[(5´-phosphoribosyl)formimino] - 5 - aminoimidazole - 4 - carboxamide ribonucleotide (5´ ProFAR) into the corresponding ribuloside and then, in association
with an ancestral G-type glutamine amidotransferase (hisH), in the
synthesis of the imidazole moiety of imidazole glycerol phosphate.
The chemical differences between the biosynthesis of the imidazole
moiety of histidine with that of purines, which is catalysed by the
ATP-dependent 5´-phosphoribosyl-5-aminoimidazole synthetase (AIR
synthetase) (Schrimshen et al., 1986), together with the absence of
189
detectable sequence similarities between AIR synthetase and the HisF
protein or the hisH-encoded glutamine amidotransferases, suggest
that the ability to synthesize imidazole evolved at least twice during
biochemical evolution.
There is experimental evidence that with high ammonia concentrations the HisH protein is not required for the in vitro synthesis
of imidazole glycerol phosphate from 5´-ProFAR with high ammonia
concentrations (Martin et al., 1971), together with recent experimental
evidence that has demonstrated prototrophic growth under high
ammonia concentrations of a Klebsiella pneumoniae strain with a mutated hisH gene (Reider et al., 1994), suggest to us the existence of an
ancestral semi-enzymatic stage, during which primitive catalytic
proteins may have been absent altogether from some of the steps of
histidine biosynthesis.
7. CONCLUSIONS AND PERSPECTIVES
The data presented here show that although all current models of
metabolic evolution acknowledge the role of gene duplication, none of
them can fully explain the structure, reaction sequence, and phylogenetic distribution of biosynthetic pathways. As shown by analysis
of ongoing genome sequencing projects, duplications have played a
major role in shaping the encoding abilities and the complexity of
cellular genomes. The discovery that homologous enzymes that
catalyse similar biochemical reactions are found in different anabolic
pathways supports the idea that enzyme recruitment took place at a
massive scale during the early development of anabolic pathways.
Evolutionary tinkering of the products of duplication events had a
major role in metabolic evolution. This possibility is supported by analysis of the available databases, which suggest that approximately 50%
of cellular genomes are the outcome of paralogous duplications that
may have preceded the divergence of the three primary domains.
Such high levels of redundancy suggest that the wealth of phylogenetic information older than the cenancestor itself may be larger
than realized, and its information may provide fresh insights into a
crucial but largely unexplored stage of early biological evolution during which major biosynthetic pathways emerged and became fixed
in a small geological timescale. Although many additional biochemical pathways evolved after divergence of the three primary
kingdoms (Chapman and Ragan, 1980), the basic genetic processes
and major molecular traits have persisted essentially unchanged for
more than 3.5 ! 109 years, perhaps owing to the linkages of the genes
190
involved and the complex interactions between metabolic routes. As
discussed elsewhere (Lazcano and Miller, 1996), such persistence
represents a striking case of conservatism that deserves further
analysis.
While it is true that the large percentage of paralogous duplications suggest that prior to the cenancestor itself simpler living systems
existed that lacked the large set of enzymes and the sophisticated
regulatory abilities of contemporary cells, the origin of the relatively
large sizes of biosynthetic enzymes has not been sufficiently analysed. Evolution of protein sizes can be understood in part by the
modular assembly of new domains and proteins, gene fusion events,
and internal duplications. However, the number of biosynthetic genes
in which duplication and fusion of ancestral modules can be recognized is surprisingly small. Two well established cases include the
HisA and HisF proteins (Fani et al., 1995) and carbamoyl phosphate
synthetase, whose large subunit also is the outome of a partial
paralogous duplication (van den Hoff et al., 1995). Additional research
is required to study if the evidence of ancient elongation events has
been eroded in other proteins, or if other mechanisms were more
important than internal duplication in the evolution of size of primitive enzymes.
The three different models of metabolic evolution discussed here
rest on an unstated assumption, i.e. the previous existence of catalytic
proteins ancestral to extant enzyme families. Recognition of the role of
ancient duplication events does not yields answers on the emergence
of the original starter types, i.e. of the enzymes that did not arise in
this manner. In some cases, the starter type may stem from slow nonenzymatic reactions where a primitive catalytic protein improved on a
previously sluggish process (Lazcano and Miller, 1996), as may have
been the case of (a) the photochemical decarboxylation of orotic acid
which yields uracil (Ferris and Joshi, 1979); (b) the ammoniadependent conversion of 5´-ProFAR into imidazole glycerol phosphate
(Martin et al., 1971); and (c) the non-enzymatic synthesis of glutamic
acid from !-ketoglutarate, ammonia, and reducing agents (Morowitz
et al., 1995).
The above examples also suggest that primitive pathways may
have existed in which only few steps were mediated by enzymes.
Additional examples of semi-enzymatic biochemical syntheses
includes a model of purine nucleotide biosynthesis based on the
ability of adenine phosphoribosyl transferase to catalyse the direct
condensation of 5-phospho-!-D-ribosyl-1-pyrophosphate with 5-amino4-imidazolecarboxamide, a key intermediate in a potentially prebiotic
191
synthesis of purines that may be analysed under laboratory conditions (Becerra and Lazcano, 1997). Testable descriptions of the
emergence of basic pathways via semi-enzymatic synthesis should
invoke not only spontaneous chemical reactions with significant
yields, but also coherent descriptions of how primitive, less specific
biological catalysts may have taken over the entire sequence of reactions. This remains an unsolved issue in all current models of the
emergence and early evolution of metabolic routes. One hundred
years after Eduard Buchner’s insight led to the recognition of the
central role that enzymes play in biological processes, we still do not
know the ultimate origin of these remarkable catalysts.
This chapter was completed during a leave of absence of A. L. as Visiting
Professor at the Universitat de València, during which the hospitality of Dr. J.
Peretó and his associates was enjoyed. Financial support of CICyT (Grant BIO960895) is gratefully acknowledged by J. P. The work of J. I. L. has been supported
in part by the Consejo Superior de Investigaciones Científicas (CSIC, Madrid,
Spain). We thank Dr. Edna Suárez for providing us with several useful
references. A. L. is an Affiliate of the NSCORT (NASA Specialized Center for
Research and Training) in Exobiology at the University of California, San
Diego.
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