trans

198 PURINES AND PYRIMIDINES
enable them to scavenge preformed purines
from the host. Thus, purine salvage serves an
indispensable nutritional function for the parasite,
and offers a plethora of potential targets
for selective therapeutic manipulation using
inhibitor approaches. These purine salvage
pathways, which will be discussed in detail,
can be quite simple, as for example in Giardia
lamblia, or myriad, intertwined, and complex,
as in Leishmania donovani.
The pathways by which pyrimidine
nucleotides are synthesized in parasites have
been less well studied, primarily because most,
although not all, parasites synthesize pyrimidine
nucleotides de novo, using essentially the
same enzymatic machinery that is found in
mammalian cells. The exceptions are the amitochondrial
protozoan parasites, Trichomonas
vaginalis, Tritrichomonas foetus, and G. lamblia,
which are obligatory scavengers of exogenous
pyrimidines. Consequently, the mechanisms
by which parasites salvage pyrimidine bases
and nucleosides tend to be less elaborate than
those for purine salvage, since pyrimidine salvage
is, with few exceptions, not essential for
parasite survival and proliferation. Additionally,
T. vaginalis and G. lamblia also appear
to lack a functional ribonucleotide reductase
(RR) activity and are thus dependent upon
exogenous sources of deoxynucleosides in
order to produce the dNTP precursors necessary
for DNA synthesis and repair (see
Table 9.1 for abbreviations).
Between 1975 and 1995, classical biochemical
approaches, particularly enzyme activity and
metabolic flux measurements, were employed
to attain an understanding of the basic enzymatic
machinery available to protozoan and
helminthic parasites for their purine and pyrimidine
metabolism. The more recent advances
in our knowledge of purine and pyrimidine
transport and metabolism in parasites,
however, originate from an interdisciplinary
amalgamation of approaches and techniques
from molecular biology, genetics, structural
biology, and immunocytochemistry. Many
genes encoding parasite purine and pyrimidine
salvage enzymes have been cloned,
overexpressed in Escherichia coli, and the
recombinant proteins purified to homogeneity
and characterized. A number of these proteins
have been crystallized and their threedimensional
structures determined in atomic
detail by X-ray crystallography. Genes encoding
purine and pyrimidine transporters also have
been isolated and functionally characterized
after expression in Saccharomyces cerevisiae,
nucleoside transport-deficient L. donovani,
and Xenopus laevis oocytes. Finally, antibodies
have been raised to a number of parasite
nucleoside transporters and salvage enzymes
and exploited to localize these proteins to subcellular
compartments. Despite these advances
in our knowledge of the mechanisms by which
parasites synthesize and acquire purine and
pyrimidine nucleotides, a complete characterization
of these pathways in any parasite has
yet to be accomplished.
Because the sequencing of several parasite
genomes is well under way, many purine
and pyrimidine salvage and interconversion
enzymes and transporters from parasites have
been identified. As the genome sequencing
projects of some parasites are completed and
others expanded or initiated, most of the genes
encoding proteins affecting purine and pyrimidine
transport and metabolism within a given
organism will be identified. The challenge will
be the functional characterization of these
genes and proteins in intact parasites. Targeted
gene replacement strategies have proven to be
informative tests of gene and protein function
in some genera.
A comprehensive review of what is currently
known on this subject is not possible within
the confines of this chapter. Rather, we will
BIOCHEMISTRY AND CELL BIOLOGY: PROTOZOA