Bariloche protein symposium argentine society for biochemistry and molecular biology

64
BIOCELL, 27 (Suppl. I), 2003
MI-P18.
THE H BOX-HARBORING DOMAIN IS KEY TO THE
FUNCTION OF THE Salmonella enterica PhoQ Mg
2+
-
SENSOR IN THE RECOGNITION OF ITS PARTNER PHOP
Castelli, M. Eugenia; Cauerhff, Ana; Amongero, Marcela; Soncini,
Fernando C.; and García Véscovi, Eleonora.
Instituto de Biología Molecular y Celular de Rosario (IBR-
CONICET), Facultad de Cs. Bioquímicas y Farmacéuticas (UNR)
and Fundación Instituto Leloir (CONICET). Rosario, Argentina.
E-mail: [e-mail]
In two-component signaling systems the transduction strategy relies
on a conserved His-Asp phosphoryl exchange between the sensor-
histidine kinase and its cognate response-regulator. Structural and
functional consensus motifs are found when comparing either
histidine kinases or response regulators present in a single cell.
Therefore, specific recognition between partners is essential to
generate the appropriate adaptive response. We dissected the Mg
2+
-
sensor PhoQ in different sub-domains and examined its interaction
with the associated response regulator PhoP. This signal
transduction system allows Salmonella to withstand environmental
Mg
2+
-limitation by triggering gene expression that is vital
throughout the infective cycle. Using resonant mirror biosensor
technology and fluorescence anisotropy we calculated the kinetic
and equilibrium binding constants and determined that the His-
phosphotransfer domain is essential for the PhoQ specific
recognition and interaction with PhoP. We also assessed the
influence of PhoP phosphorylation in the partners interaction which
supports the physiological significance of PhoQ phosphatase
activity. Moreover, we showed the role of the His-phosphotransfer
domain in the bimolecular transphosphorylation and provide
evidence that this region undergoes dimerization.
MI-P19.
IDENTIFICATION OF ESSENTIAL AMINO ACID
RESIDUES IN THE BRUCELLA ABORTUS CYCLIC
GLUCAN SYNTHASE CGS
Ciocchini, Andrés E.; Briones, Gabriel; Iñon de Iannino, Nora;
Ugalde, Rodolfo A.
IIB-INTECH, UNSAM-CONICET, Buenos Aires, Argentina. E-
mail: [e-mail]
Cgs is an integral inner membrane protein of 316 kDa involved in
the synthesis of cyclic 
β-(1,2)-glucan by a novel mechanism  in
which the enzyme acts itself as protein intermediate.  Cgs uses
UDP-glucose as donor-sugar and has the three-enzymatic activities
required for the synthesis of the polysaccharide: initiation,
elongation and cyclization. The first one may catalyze the transfer
of the first glucose from UDP-Glc to an unknown amino acid of
the protein intermediate. The second enzymatic activity [UDP-
Glc:
β-(1,2) oligosaccharide glucosyltransferase] may be
responsible for chain elongation. Finally, the third activity may
catalyze glucan cyclization and release from the protein. By
comparing the sequence of Cgs to those of the members of
glycosyltransferase family 2 (GTF2), we identified the
D
1
,D
2
,D
3
,(Q/R)XXRW motif that is highly conserved in Cgs and
in the putative active site of numerous processive 
β-
glycosyltransferases. By site-directed mutagenesis we found that
replacement of Asp-636 (D2) by asparagine abolished the enzyme
activity  in vitro and in vivo. Single substitutions of each of the
amino acids of the RXXRW motif at positions 782, 785 and 786
with alanine resulted in the loss of enzyme activity in vitro.  In
vivo production of cyclic glucan was affected in the R785A and
W786A mutants but not in the R782A mutant. These results
indicate that Asp-636, Arg-785 and Trp-786 are essential for Cgs
activity and may be implicated in its elongation activity.
MI-P20.
THE OXIDANT-RESPONSIVE DIAPHORASE OF
RHODOBACTER CAPSULATUS IS A  FERREDOXIN
(FLAVODOXIN)-NADP(H) REDUCTASE
Bittel, Cristian; Tabares, Leandro C.; Medina, Milagros
∗ and
Cortez, Néstor.
IBR (CONICET & UNR) Suipacha 531, S2002LRK-Rosario and
∗Dto. de Bioquímica y Biol. Molecular, Universidad de Zaragoza,
Spain. E-mail: [e-mail]
Challenge of Rhodobacter capsulatus cells with the superoxide
propagator methyl viologen results in the induction of a diaphorase
activity identified as a member of the ferredoxin (flavodoxin)-
NADP(H) reductase family (FPR) by N-terminal sequencing. The
enzyme was purified 400-fold to apparent homogeneity using ion
exchange chromatography as a monomer of 30 kDa. The fpr gene
was cloned and expressed in Escherichia coli, rendering a product
with essentially the same properties as that isolated from the
photosynthetic bacterium. Both native and recombinant forms were
able to bind and reduce Rhodobacter flavodoxin (NifF) and to
mediate typical FPR activities such as the NADPH-driven
diaphorase and cytochrome c reductase reactions.
In  E. coli, and presumably also in Salmonella  and related
enterobacteria, FPR provides reduced Fd or Fld for a number of
reactions involved in amino acid and nucleotide metabolism, biotin
synthesis and iron-sulphur cluster assembly. In nitrogen-fixing
bacteria such as Azotobacter and Rhodobacter species, ferredoxins
and flavodoxins are the immediate electron donors to the
nitrogenase. Steady-state and stopped-flow kinetic measurements
on the recombinant Rhodobacter FPR rendered slower reaction
rates than the plastidic enzyme, although compatible with electron
currents through nitrogenase.
MI-P21.
IN VIVO ERYTHROCYTE FUSION IN MICE WITH
EXPERIMENTAL CHAGAS DISEASE
Cossy Isasi S., Pereira B.M.I., Rodríguez M. and Bronia D.H.
Cátedra de Bioquímica y Biología Molecular, Fac. Cs. Médicas,
UNC. E-mail: [e-mail]
We study molecular aspects of Trypanosoma cruzi (Chagas disease
agent) mammalian host cell interaction .We first described that
parasite induced normal erythrocyte fusion in vitro. The lipidic
profile, the increase in parasite phospholipid turnover, the
inhibition with parasites cultured in ganglioside supplemented BHI
medium and anti-PLA2 antibodies recognition of parasite proteins
suggested a parasite PLA2 involvement in the interaction with
host cells. Decreased hydrolysis of fluorescent and radioactive
substrate (25%) and in vitro fusion (50%) when gangliosides  were
incorporated to substrate or gangliosides  or anti-PLA2  antibodies
were incorporated at the beginning of the assay to the incubation
medium, points to a parasite PLA2 structurally homologous to
mammalian PLA2 with extracellular location. From these results
erythrocyte fusion seemed to be a good model to evidence PLA2
activity. Now we report that fusion of red blood cells occurs in
vivo. Fusion bodies were observed in all blood samples of T.cruzi
infected mice immediately after extraction with  heparine at 21ºC,
no matter of parasite strain an origin, mouse blood or media
supernatant of nurse cells with or without freezing.

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