trans

220 PURINES AND PYRIMIDINES
in Plasmodium-infected erythrocytes. Thus,
Plasmodium is capable of making pyrimidines
de novo. The enzymes involved in the pathway
have not been well studied but seem functionally
akin to their mammalian counterparts.
The six enzymes in the de novo UMP pathway,
however, are all distinct and not components
of multifunctional enzymes; and PfDHODH
appears to be mitochondrial. Metabolic studies
have confirmed the presence of CTPSII,
RR, and TS activities. TS is part of a DHFR–TS
complex, as it is for most protozoan parasites.
Pyrimidine salvage by P. falciparum and other
Plasmodium species is minimal or non-existent.
Any uracil salvage that is observed can theoretically
be ascribed to PfOPRT of the de novo pathway,
an enzyme that recognizes uracil poorly.
Toxoplasma gondii
T. gondii also synthesizes pyrimidine nucleotides
de novo. The T. gondii CPSII gene has
been cloned and disrupted, and mutants at
the CPSII locus are auxotrophic for pyrimidines.
These pyrimidine auxotrophs are completely
avirulent in mice and are able to evoke
long-term protective immunity to toxoplasmosis.
Thymidylate nucleotides are synthesized
from dUMP via DHFR–TS. All pyrimidine
salvage in T. gondii proceeds through uracil,
which is salvaged to the nucleotide level
by uridine phosphoribosyltransferase (UPRT).
Salvage is not essential for parasite viability,
since UPRT-deficient mutants have been made
and exhibit the same growth rate as wildtype
parasites. These mutants, unlike wild-type
parental cells, fail to take up uracil, uridine, and
deoxyuridine. Neither wild-type nor UPRTdeficient
T. gondii take up other pyrimidine
bases or nucleosides, including cytosine,
thymine, cytosine, deoxycytidine, and thymidine.
Thus UPRT is the only enzyme in T. gondii
that can salvage preformed pyrimidines. The
TgUPRT gene has been cloned by insertional
mutagenesis, and the recombinant protein
has been produced in bulk and its crystal structure
solved to 1.93 Å.
Eimeria tenella
E. tenella sporozoites can incorporate
[ 14 C]aspartate and [ 14 C]orotate into pyrimidine
nucleotides and thymidylate through
DHFR–TS, so a pyrimidine biosynthetic pathway
must be present. The parasite can also
salvage certain pyrimidines including uracil,
uridine, and cytidine, but not thymidine. The
biochemistry of E. tenella pyrimidine enzymes
has not been studied.
Kinetoplastida
Leishmania
The ability of Leishmania to carry out pyrimidine
biosynthesis de novo was initially established
from growth studies that indicated that
exogenous pyrimidines were not needed for
parasite proliferation. All of the pyrimidine
biosynthetic enzymes were subsequently
detected in a multiplicity of species. OPRT and
ODC were shown to be biochemically separable
enzymes, both of which are located in the
glycosome, and DHODH is not associated
with the parasite mitochondrion. Also, the
DHFR and TS activities were shown to be part
of a bifunctional DHFR–TS protein that has
been well characterized at the biochemical
and structural level.
Pyrimidine salvage and interconversion in
Leishmania is far less complex than purine
salvage. Orotate can be taken up through OPRT
and distributed among UTP and CTP. Thymidine
is incorporated inefficiently, a process that
appears to be TK-mediated, since thymine is
not taken up. Uridine cleavage, dCD, and UPRT
BIOCHEMISTRY AND CELL BIOLOGY: PROTOZOA