trans

PURINE METABOLISM 211
is not currently present in the database. PfADA
appears to differ from its human counterpart
by its refractoriness to erythro-9-(2-hydroxy-
3-nonyl)adenine. It is unclear whether the
parasite has a functional APRT activity. It is
possible that the gene has yet to be discovered
or that the early biochemical evidence for the
existence of the enzyme was influenced by the
presence of AD activity. The ability of P. falciparum
to salvage adenine, however, intimates
that either APRT or an AD/HGXPRT pathway
must be operative. PlasmoDB also contains
DNA sequences encoding ASS, ASL, IMPDH,
GMPS, AMPD, and GMPR. Metabolic flux
measurements prove the functionality of the
first four of these six enzymes, but none of the
six has been studied in detail. The current
model for purine salvage in P. falciparum is
depicted in Figure 9.4.
Toxoplasma gondii
T. gondii also cannot synthesize purine
nucleotides de novo but can proliferate without
added purine. Thus, it can derive all its
purine needs from the host cell. Early studies
suggested that host adenylate nucleotides
could access the parasitophorous vacuole and
be dephosphorylated by the tachyzoite, the
actively proliferating stage of the parasite.
AMP appeared to be the most efficiently utilized
nucleotide, although both ATP and ADP
were also incorporated. The vacuole itself contains
high concentrations of NTPase (apyrase)
activity that is capable of hydrolyzing ATP to
ADP and AMP. A number of different isoforms
of the NTPase were discovered and shown to
exhibit slightly different substrate selectivities.
Despite this finding, T. gondii lacks a 5-NT
activity that would dephosphorylate AMP to
adenosine and allow purine entry into the cell.
Furthermore, a nucleoside transport-deficient
T. gondii strain was greatly compromised in
AMP-uptake capability. This implies that AMP
was dephosphorylated to adenosine by contaminant
host 5-NT activity. These data argue
against a role of NTPase in purine acquisition
by the parasite.
Extracellular tachyzoites are capable of taking
up a number of purines including adenosine,
inosine, guanosine, adenine, hypoxanthine,
guanine, and xanthine. Adenosine was incorporated
at a rate 10–25-fold greater than any of
the other purines, suggesting that adenosine
was the primary nutritional purine for the parasite.
Biochemical studies further revealed that
AK is the most active purine salvage enzyme
present in tachyzoite extracts, more than
ten-fold more active than any other enzyme
(Figure 9.5). No other nucleoside kinase or
phosphotransferase activities were detected.
T. gondii extracts were also capable of cleaving
guanosine and inosine, but not adenosine,
implying that PNP activity was present. Both AD
and GD activities were found, as were phosphoribosylating
activities for all four bases.
However, the observed adenine incorporation
is likely imputed to AD-mediated deamination
and subsequent hypoxanthine salvage through
TgHGXPRT. Interestingly, radiolabeled inosine,
hypoxanthine, and adenine labeled both
adenylate and guanylate nucleotides, while
guanine, guanosine, and xanthine only labeled
guanylate nucleotide pools. These data imply
that T. gondii lacks GMPR activity, although all
the branchpoint pathways from IMP to AMP
and GMP must be intact (Figure 9.5).
The genes encoding TgAK and TgHGXPRT
have been cloned using insertional mutagenesis
strategies, and both recombinant proteins
have been purified and characterized. TgAK is
specific for adenosine, while TgHGXPRT recognizes
hypoxanthine, guanine, and xanthine,
although xanthine binds less well than the other
bases. High resolution crystal structures for
both TgAK and TgHGXPRT have been obtained,
BIOCHEMISTRY AND CELL BIOLOGY: PROTOZOA