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EVOLUTION
OF BIOLOGICAL MOLECULAR ENTITIES BY DUPLICATION
Introduction
The
aim of the project
State
of the art
- genome duplications
- deoxyribonucleoside kinases
- de novo /
catabolic biochemical pathways
References
Introduction
Many of the genomes
currently sequenced, as from the yeast
Saccharomyces cerevisiae, the nematode worm Caenorhabditis
elegans, the fruit fly Drosophila
melanogaster, the thale cress Arabidopsis
thaliana, the human Homo sapiens,
and bacteria such as Escherichia coli
and Bacillus subtilis, show a high
degree of sequence redundancy. Up to half of the genes present within the
genomes are not unique and are a result of duplication at some point during the
evolutionary history (17, 18).
Gene duplication is
believed to play one of the major roles in biological evolution (16). Two genes
that are derived from a gene duplication are said to be paralogous, and two
genes in two different species are orthologous if they were derived from the
same gene through speciation. In most organisms genes are continuously
duplicated generating paralogues, and often afterwards one of the copies is
removed from the genome. However, when a set of parallel genes is created, gene
copies can be functionally specialised through accumulation of mutations and
afterwards permanently preserved in the genome. Preservation can be achieved
either by origin of a new function by one of the duplicated genes, or by
partitioning of the ancestral function(s) between the two duplicated genes. Gene
duplications can occur as single-gene duplications, duplications of short
chromosomal segments, duplications of single chromosomes or by duplications of
the entire genome. Even a limited number of
gene duplications can have a global effect on the cell physiology and its
proteosome and metabolome.
The
aim of the project
Our group would like to
understand what was the impact of duplications on the origin of modern genes,
chromosomes, genomes, enzymes, biochemical pathways, and novel styles of life,
such as anearobic and aerobic mode of existence. For this purpose, a model for
each of the previously mentioned elements has been chosen and studied in a
variety of eukaryotic organisms. We study the whole genome duplication in yeasts
as a background for development of the anaerobic and fermentative life style,
diversity and function of deoxyribonucleoside kinases in eukaryotes to
understand enzyme duplication and specialisation, anabolic and the corresponding
catabolic enzymes and pathways to understand interconversion of the enzyme
function and the corresponding biochemical pathways, and development of
multi-domain enzymes by fusion of single-domain proteins.
State
of the art
genome duplications
In the genomes of the
fruit fly, the nematode worm and the fission yeast Schizosaccharomyces
pombe, most paralogous genes are dispersed, apart from a few small clusters
of duplicated genes. By contrast, the arrangement of duplicated genes in the
plant A. thaliana and the baking yeast
S. cerevisiae, shows large segmental
duplications (20, 21). In these two organisms, members of homologous gene pairs
are in the same order along two distinct segments that are sporadically
interspersed by unique genes. The app. 100 duplicated segments in A.
thaliana originated from four different
large-scale duplication events, followed by diversification of some of
the duplicated genes and concurrent loss of many others (20). These gross
duplications could represent the basis for diversification among angiosperm
plants. A similar but single whole-genome duplication could also have occurred
at the root of the vertebrate providing
the basis for radiation of different vertebrate lineages (19).
Several sequencing
projects on different yeast species provide an excellent model to understand the
role of genome duplications in evolution of different groups of organisms. It is
now clear that the situation with large duplicated regions in the genome
of Saccharomyces
yeasts is rather unique among yeasts. A single genome duplication was initially
proposed as the origin of the current S.
cerevisiae duplicated regions (21). However, a series of continuous, smaller
duplications could also result in a similar genome configuration (6, 17).
In our recent studies
we showed that extensive reorganisation of the chromosomes took place upon
separation of different Saccharomyces
species (9, 12). The degree of synteny among various yeast species decreases as
the phylogenetic distance increases. In addition to the shuffling of fragments,
insertions and deletions of single genes, gene duplications played a significant
role in the evolution of Saccharomyces
chromosomes (6, 12, 17). What could be nature’s rationale for the yeast genome
duplications? We have proposed that the origin of duplications coincides with
the origin of the ability to grow under anaerobic conditions. This step
definitely required a major remodelling of the yeast metabolism and regulatory
mechanisms. Althrough the majority of yeast cannot grow in the absence of
oxygen, at least two groups, Saccharomyces
and Brettanomyces/Dekkera,
independently developed the ability to grow under anaerobic conditions (13).
Apparently, the anaerobic life style of Saccharomyces
yeasts coincides with the appearance of larger quantities of free mono-sugars
originating from the fruits of higher plants. This event coincides with the
radiation of the modern plants app. 200 million years ago and overlaps with
plant whole genome duplications. The question is if there has been only one
gross duplication in the Saccharomyces
genome or a series of smaller duplication? The present data are still not
sufficient to distinguish between the two alternatives and further analysis is
necessary. However, a large amount of sequencing data, including several Saccharomyces
species may be completed by the end of the year. At present we have already
analysed a number of genes, homologous to the S.
cerevisiae duplicated genes to get an idea about the origin of the yeast
duplication(s).
deoxyribonucleoside kinases
Deoxyribonucleoside
kinases catalyse the phosphorylation of deoxyribonucleosides (dN) to the
corresponding deoxyribonucleoside monophosphates (dNMP). They are the key
enzymes in the salvage of deoxyribonucleosides originating extracellularly or
from intracellular breakdown of DNA (2). Subsequently, dNMPs are phosphorylated
into diphosphates (dNMP) and triphosphates (dNTP), which are direct precursors
of DNA. In humans, the salvage pathway is extremely important for activation of
several anti-viral and anti-cancer drugs.
Deoxyribonucleoside
kinases exhibit a lot of diversity among the analysed organisms. Mammals have
four deoxyribonucleoside kinases with overlapping specificities: cytoplasmic
thymidine kinase 1 (TK1) phosphorylates only thymidine, mitochondrial thymidine
kinase 2 (TK2) phosphorylates thymidine and deoxycytidine, cytoplasmic
deoxycytidine kinase (dCK) phosphorylates deoxyadenosine, deoxyguanosine and
deoxycytidine, and mitochondrial deoxyguanosine kinase (dGK) phosphorylates only
purine dNs, deoxyadenosine and deoxyguanosine (2). In insects, only one
deoxyribonucleoside kinase, (dNK), capable of phosphorylating all four natural
substrates, was discovered by our group (14, 15).
The sequences of
mammalian and insect deoxyribonucleoside kinases can easily be aligned
pointing out that they originated from a common progenitor kinase by a series of
gene duplications. Deoxyribonucleoside kinases are a very illustrative example
of preservation of duplicated genes
by partitioning of the ancestral enzyme functions. So far the timing of
duplications is unclear. However, pressumably the first duplication generated
the progenitor of TK1-like kinases and the progenitor of TK2-, dCK/dGK-like
kinases. The later progenitor could have a broad substrate specificity, which
was after further duplications narrowed in different kinase lineages, leading to
the modern enzymes with limited substrate specificity. When the 3D structures of
different kinases were compared, the key amino acid residues, determining the
substrate specificity were identified (10, 11). The site-directed mutagenesis of
these sites provided mutant enzymes with changed substrate specificity. A
similar scenario, based on a limited number of single amino acid changes, could
have operated during the evolutionary history of deoxyribonucleoside kinases.
While the amino acid sequences among deoxyribonucleoside and deoxyribonucleoside
monophosphate kinases do not show siginficant identity, the structural studies
suggest that these two groups of enzymes have a common origin. Studies of
kinases in different groups of organisms is still necessary to get a more
detailed understanding of the evolutionary history. It may be that one of the
duplications was a part of the whole-genome duplication at the origin of
vertebrate lineages.
de novo /
catabolic biochemical pathways
Central metabolites,
which are synthesized in the cell, can often also be degraded in the same cell.
An example are pyrimidine bases, which are synthesized de novo in all eukaryotes,
and in a majority they can also be degraded. This catabolic pathway is of
crucial importance in cancer patients, because it degrades several
chemoterapeutic drugs (5). Three enzymes are involved: dihydropyrimidine
dehydrogenase (DPDase), dihydropyrimidine amidohydrolase (DHPase) and
beta-alanine synthase (BSase). Our work on yeast and other eukaryotes has
recently shown that these catabolic enzymes highly resemble dihydroorotate
dehydrogenase (DHODase), dihydroorotase (DHOase), and aspartate transcarbamylase
(ATCase), respectively, which catalyse the fourth, third and second step of the
de novo pyrimidine pathway (7, 8). This is not surprising because in
essence the anabolic and the corresponding catabolic enzyme catalyse the same
reaction, only in the opposite direction. Apparently, at some point in the
evolution the genes belonging to the anabolic pathway were duplicated and
acquired a new function. For example, when the gene for DHOase, which
pressumably catalysed a reversible reaction, was duplicated, one of the copies
got specialised into anabolic and another into catabolic enzyme (7).
Comparing homologous enzymes involved in metabolism of pyrimidines from
different organisms, it seems that the switch between the anabolic and catabolic
functions occurred frequently during the evolutionary history. It seems that
organisms can duplicate a gene coding for a certain enzyme, or even the whole
biochemical pathway, and afterwards one of the copies is modified to acquire a
new function. We are interested to know about the timing of these events, the
reaction mechanism of homologous enzymes, as well as we would like to
experimentally mimic the conversions between different functions.
Another interesting
aspect is the origin of multi enzymatic activities encoded by a single
polypeptide chain. In general, in prokaryotes the enzymes are small, catalysing
only one reaction. On the other hand, in eukaryotes enzymes are often composed
of several covalently linked domains, which can catalyse several enzymatic
activities. Such multi-functional enzymes have originated by the fusion of genes
encoding mono-functional proteins.
Among pyrimidine
metabolising enzymes, two examples of multi-functional enzymes can be found. In
mammals, the first three reactions of de novo pyrimidine pathway are catalysed
by a single CAD enzyme carrying the catalytic activities of carbamoylphosphate
synthetase (CPSase), ATCase and DHOase (4). In yeast and other fungi, the URA2
enzyme carries only CPSase and ATCase activities and a “silent” DHOase
domain, while DHOase activity resides within a separate enzyme (4). Thus in some
yeasts three DHOase-like copies are expected: a silent DHOase in the
multi-enzymatic URA2 protein, an anabolic copy and a catabolic, DHPase, copy. In
bacteria all three activities are carried out by mono-functional domains.
Therefore, the fusion of mono-functional domains occurred before the fungi
lineage branched out from the animal lineage. Similarly, the first pyrimidine
catabolic enzyme, DPDase, is in mammals a large protein displaying five
structural and functional domains (5). However, our recent results point out
that in bacteria, plants and yeast the DHPase activity is catalysed by separate
protein sub-units encoded by several independent genes. Apparently, the fusion
of the mono-functional proteins happened only in the animal lineage. The
corresponding enzymes from several groups of organisms should now be analysed to
provide a further insight into the evolutionary history of multi-functional
enzymes. Often some of the mono-functional units participating in the origin of
multi-functional enzymes had just been duplicated prior to the fusion event.
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