trans

PURINE METABOLISM 209
purine available to the cell is in excess of its
requirements. Urate is synthesized from
hypoxanthine and xanthine, an oxidation
reaction that is catalyzed by XO. XO is absent
in both protozoan and helminthic parasites.
Because of the nutritional necessity of purines
and the relative scarcity of purines in the environment,
it is unlikely that parasites would
have a great need for eliminating dispensable
purine.
The purine deoxynucleotide substrates for
DNA synthesis are derived from ribonucleotides.
Most RRs are two-subunit enzymes
that catalyze the reduction of the 2-hydroxy
moiety of all four naturally occurring purine
and pyrimidine ribonucleoside disphosphates
to the corresponding deoxynucleotides. RR is
the only known mechanism for de novo production
of deoxynucleotides, and the enzyme
is subject to complex allosteric regulatory
mechanisms that ensure a balanced supply of
all four dNTPs for DNA synthesis. RR activity
in mammalian cells is also cell-cycle dependent;
the enzyme is active during the S phase
of the cell cycle when DNA is replicated and
relatively inactive during the quiescent
(G1 and G2) and mitotic (M) phases of the cell
cycle. Deoxynucleotides can also be generated,
albeit inefficiently, via kinase enzymes.
AK, dCK, and several mitochondrial deoxynucleoside
kinases can metabolize deoxyadenosine
and/or deoxyguanosine to the nucleotide
level. Most parasites are thought to generate
deoxynucleotides directly via ribonucleotide
reduction. The existence of RR is supported
by the ability of parasites to grow in medium
lacking deoxynucleosides, to incorporate radiolabeled
bases and nucleosides into DNA, the
detection of RR activities in crude parasite
lysates, the existence of parasite clones encoding
RR subunit homologs, and RR subunit
sequences in the parasite genome sequencing
databases.
Apicomplexa
Plasmodium
Because infection of erythrocytes with Plasmodium
spp. dramatically augments their capacity
to salvage purines, most of the early knowledge
on purine salvage capabilities of malarial
parasites was gleaned from comparisons of
uninfected and infected erythrocytes. Like the
mammalian red blood cell, Plasmodium species
cannot synthesize purines de novo, and their
growth is contingent on salvage. Any of a number
of purines, including hypoxanthine, guanine,
adenine, inosine, and adenosine, can
satisfy the purine requirements of P. falciparum.
Hypoxanthine is likely the preferred source of
malarial nucleotides, and can be acquired
directly from blood or from erythrocytes
through nucleotide degradation. Furthermore,
addition of XO, which converts hypoxanthine
to urate, to the growth medium inhibits P. falciparum
proliferation, strongly implying that
extracellular hypoxanthine is the critical source
for parasite nucleotides. ADA and PNP activities
necessary for adenosine deamination and
inosine cleavage are also present in both the
red blood cell and parasite and are likely major
contributors to purine salvage as well (Figure
9.4). Similar findings were obtained with rodent
and avian malarias. Because a single purine can
fulfill all of the parasite’s nucleotide requirements,
the enzymatic machinery to convert
IMP to adenylate and guanylate nucleotides
must be operational in the parasite.
A number of purine salvage and interconversion
enzymes from P. falciparum have been
characterized at the biochemical level (Figure
9.4). Most prominent among these are HGX-
PRT, PNP, and ADA. The PfHGXPRT gene has
been cloned, sequenced, and overexpressed in
E. coli, and the recombinant protein purified
to homogeneity. Preferred substrates for
BIOCHEMISTRY AND CELL BIOLOGY: PROTOZOA