Biology Reference
In-Depth Information
derivatives), the TbrPDEB1 and TbrPDEB2 genes have undergone a partial gene
conversion, exchanging very precisely the GAF-A domain of TbrPDEB2 with the
corresponding domain of the upstream gene TbrPDEB1. Thus, all these strains are
heterozygous for TbrPDEB2, carrying one allele of TbrPDEB2a (the wild-type
version) and one allele of TbrPDEB2b (with a GAF-A domain that originates
from TbrPDEB1). While this gene conversion has no functional consequences, it
provides a convenient genetic tool for unambiguous identification of trypanosome
strains (Kunz et al.
2009a
).
Both enzymes are cAMP-specific, with
K
m
values in the order of 5
m
M (Zoraghi
and Seebeck
2002
; Rascon et al.
2002
; Seebeck et al. unpublished data). The GAF-
A domains of both enzymes bind cAMP and cGMP with high affinity (
K
D
cAMP
16 nM;
K
D
cGMP 275 nM; Laxman et al.
2005
; Seebeck et al. unpublished data),
suggesting that the domains might function as highly sensitive cyclic nucleotide
sensors. Between the two GAF domains is a predicted PKA phosphorylation site
(K
387
-S
390
). This site is conserved in all kinetoplastid PDEB1 and PDEB2 enzymes
and may be functional. At the C-terminus of TbrPDEB1, residues S
920
,S
922
, and
S
923
have been identified in the
T. brucei
phosphoproteome, while none of the
corresponding residues of TbrPDEB2 are represented (Nett et al.
2009
).
The two PDEs have also been investigated in
T. cruzi
[TcrPDEB1 (Diaz-Benjumea
et al.
2006
) and TcrPDEB2 (D'Angelo et al.
2004
)] and shown to have very similar
characteristics. The mRNAs for both enzymes are present in all three developmental
stages (amastigotes, trypamastigotes, and epimastigotes). Both enzymes are cAMP-
specific, with
K
m
values of 2.8
m
M (TcrPDEB1 (Diaz-Benjumea et al.
2006
) and
7.8
m
M (TcrPDEB2 (D'Angelo et al.
2004
). Interestingly, the fluorescent cAMP
derivative MANT-cAMP is hydrolyzed with a
K
m
very similar to that of cAMP [1.8
vs. 2.8
m
M (Diaz-Benjumea et al.
2006
)]. An analysis of cyclic nucleotide binding to
the GAF-A domains of the
T. cruzi
enzymes revealed significant differences
between the
T. brucei
homologues: cAMP bound to the GAF-A domain of
TcrPDEB1 with a
K
D
of 970 nM and the corresponding values for TcrPDEB2
were 190 nM (by filter binding) or 520 nM (by isothermal microcalorimetry). The
GAF-A domain of TcrPDEB1 did not measurably bind cGMP, while the GAF-A
domain of TcrPDEB2 bound cGMP with a
K
D
of 2.5 mM (Diaz-Benjumea et al.
2006
). Further experimentation is required to resolve whether these low binding
affinities of the
T. cruzi
GAF-A domain are in fact genuine.
PDEC
. All kinetoplastid genomes contain one copy of PDEC. The predicted
proteins (about 900 amino acids) are unusual in that they contain a FYVE-related
domain (Kutateladze
2007
) at the N-terminus, followed by a coiled-coil region. This
architecture suggests that the enzymes might be located in the endosomal compart-
ment. The catalytic domain is situated approximately in the middle of the polypeptide
and is followed by a rather long C-terminal region (298 amino acids in TbrPDEC).
TcrPDEC from
T. cruzi
was independently characterized by two groups (Kunz
et al.
2005
; Alonso et al.
2006
), who obtained different values for its specificity.
While Alonso et al. (
2006
) reported that the enzyme is cAMP-specific (
K
m
20
m
M),
Kunz et al. (
2005
) found that it hydrolyzes both cAMP (
K
m
30
m
M) and cGMP (
K
m
78
m
M) with similar
V
max
. The reason for this discrepancy remains unresolved.
