trans

PURINE METABOLISM 213
FIGURE 9.6 Kinetoplastid purine salvage and interconversion pathways. The white line indicates that AD enzyme
(1) is present in Leishmania promastigotes but not in T. cruzi or T. brucei. Enzyme identities are listed in Table 9.1.
PRT genes have been cloned, and the recombinant
proteins have been purified and characterized.
LdHGPRT and LdAPRT exhibit
absolute specificity for the purines for which
they are named, whereas LdXPRT, for which
xanthine is the preferred substrate, can also
recognize hypoxanthine and guanine. Pyrazolo
(3,4-d)pyrimidine bases, e.g. allopurinol and
novel antileishmanial compounds that are nontoxic
to mammalian cells, require HGPRT
for their conversion to toxic phosphorylated
metabolites, but allopurinol is still a relatively
inefficient LdHGPRT substrate. LdHGPRT
and LdXPRT also contain a COOH-terminal
tripeptide that is a topogenic signal for targeting
proteins to the glycosome, a unique kinetoplastid
organelle. Both LdHGPRT and
LdXPRT have been localized to the glycosome
by immunofluorescence, immunoelectron
microscopy, and subcellular fractionation.
Conversely, LdAPRT is a cytosolic enzyme.
Other L. donovani enzymes that can convert
purines to the nucleotide level are LdAK and
possibly LdNPT. LdNPT has been implicated
in the metabolism to the nucleotide level of
the antileishmanial pyrazolopyrimidine nucleoside
analogs, allopurinol riboside, formycin
B, and 4-thiopurinol riboside, but whether the
enzyme recognizes naturally occurring purine
nucleosides is unknown.
Gene replacement studies have shown that
ldhgprt, ldaprt, and ldxprt knockouts are
all viable as promastigotes, proving that none
of the three LdPRTs is by itself essential for
parasite growth. LdAK is also non-essential,
since AK-deficient L. donovani has been created
after mutagenesis. Combinations of mutations
at the LdHGPRT, LdAPRT, and LdAK loci
have revealed that even a triple mutant
deficient in all three activities can survive on
any purine nucleobase or nucleoside tested.
Further gene replacement experiments have
failed to generate a double ldhgprt/ldxprt
knockout providing powerful, but only suggestive,
negative evidence that L. donovani
promastigotes require either a functional
HGPRT or XPRT activity for growth.
The PRPP and nucleobase substrates for
PRT enzymes are derived from PS and NHs,
respectively. L. donovani has at least three NH
activities that cleave nucleosides to their corresponding
bases and ribose: one specific for
inosine, uridine, cytidine, and xanthosine
(IUNH); one for deoxynucleosides; and one
for inosine and guanosine. The IUNH from L.
major has been cloned, and the recombinant
BIOCHEMISTRY AND CELL BIOLOGY: PROTOZOA