trans

214 PURINES AND PYRIMIDINES
protein evaluated for substrate selectivities
and affinities. Moreover, a 2.5 Å crystal structure
has been determined. The IUNH appears
to be non-essential, because Dldiunh knockouts
are viable as promastigotes. L. donovani
also possesses an AP activity that cleaves
adenosine to adenine. To produce NH substrates
from nucleotides and nucleic acids,
L. donovani has surface membrane 3 NT/
nuclease and 5 NT activities, as well as an active
surface phosphatase activity. The L. donovani
3 NT/nuclease and acid phosphatase genes
have been cloned, and their proteins localized
and studied in detail.
Metabolic flux experiments with L. donovani
promastigotes have demonstrated that
adenine and inosine are funneled to hypoxanthine
through AD and NH, respectively, whereas
guanine is converted to xanthine by GD. Moreover,
mutationally derived APRT-deficient
L. donovani take up adenine as efficiently as
wild-type cells. Thus, APRT does not appear
to play an important role in purine uptake
by promastigotes. A more important role for
APRT can be inferred from studies with
amastigotes, which lack AD, but possess
ADA, activity. This implies that adenine is
salvaged via APRT in amastigotes, whereas
adenosine can either be deaminated to inosine
which is then cleaved by NH or phosphorylated
directly by AK. AK also plays a
prominent role in adenosine uptake by the
promastigote, since AK-deficient parasites
do not take up adenosine as efficiently as
parental cells.
The ability of the parasite to interconvert
adenylate and guanylate nucleotide pools
demonstrates that GR and AMPD are both
expressed in L. donovani. The LdGR can be
distinguished from its human counterpart by
several kinetic features and by its increased sensitivity
to inhibition by pyrazolopyrimidine
nucleotides. The LdASS is also unique in that
it can accept allopurinol ribonucleotide
monophosphate and formycin B monophosphate
as substrates. The unique properties
of LdASS and LdGR likely contribute to the
selective actions of pyrazolopyrimidines on
the parasite.
Trypanosoma cruzi
Purine metabolism in T. cruzi is quite similar
to that in Leishmania with few exceptions
(Figure 9.6). T. cruzi epimastigotes and amastigotes
incorporate bases and nucleosides into
their nucleotide pools, and adenylate and
guanylate nucleotides are interconvertible.
Bases are preferred over nucleosides. The epimastigotes
appear to phosphorylate adenosine
through AK, although the enzyme has not
been isolated, and the PRT activities appear to
be similar to those of L. donovani, although
only TcHGPRT has been well studied at the
molecular level. Elegant structural studies
promoting TcHGPRT as a target for inhibitor
design have been accomplished, although
whether TcHGPRT is a good target could be
complicated by XPRT- and APRT-mediated
purine salvage. Whether TcHGPRT is essential
awaits the construction of a tchgprt null
mutant. There have been conflicting reports
over whether T. cruzi epimastigotes express
AD activity. Thus, adenine may be phosphoribosylated
directly to AMP or deaminated to
hypoxanthine, depending on whether APRT
or AD is most active. Epimastigotes appear
to have AK activity, and a TcAK cDNA is present
in the T. cruzi database. T. cruzi also
expresses purine nucleoside-cleaving enzymes,
including a phosphorylase, an inosine/guanosine
NH, and a 2-deoxyinosine NH. Finally,
the T. cruzi ASS and ASL proteins are akin
to their leishmanial equivalents. Overall, the
purine salvage pathway of T. cruzi is similar to
that of L. donovani, except that the substrate
BIOCHEMISTRY AND CELL BIOLOGY: PROTOZOA