Laboratory
Page
Dr. Mario L. Calcagno 
calcagno@bq.unam.mx
Protein engineering applied to the study of enzyme
function
SUMMARY
The study of the relation between enzyme structure and function is a very active
research field at this time. Our laboratory is researching the structural bases
of enzyme functioning using as experimental model some soluble enzymes from the
amino sugar utilization pathway, principally human recombinant and Escherichia
coli glucosamine-6-phosphate deaminase (E.C. 5.3.1.10) and N-acetylglucosamine
6-phosphate deacetylase (EC 3.5.1.25) from E. coli. The laboratory's main
interest is to understand the catalytic mechanism of these enzymes and, particularly,
to reveal the dynamics and allosteric transition mechanism of glucosamine-6-phosphate
deaminase. This enzyme reversibly converts glucosamine 6-P into fructose 6-P and
is activated by N-acetylglucosamine 6-P. The enzyme is a hexamer with six
equal subunits whose structure has been resolved by crystallographic methods (see
cited publications). The study of this enzyme's function in our laboratory is
approached using very different methodologies, including directed mutagenesis,
specific chemical modification (chemical mutation) and physico-chemical, kinetic
and structural studies of the constructed mutants. The programmed modification
of the wild molecules of these enzymes is complemented with the study of natural
variants, especially the different type of allosteric regulation found in the
deaminases of different organisms. The construction of site-specific mutants has
allowed us to obtain new deaminases with different allosteric behavior, for example
mutants of the bacterial enzyme whose regulation is similar to a mammal's enzyme.
This study is providing us with a great deal of information on the dynamics of
the allosteric transition, its different modalities and evolutionary adaptations.
PARTICIPANTS
COLLABORATIONS:
- Dr. Myriam M. Altamirano, (protein engineering and design);
- Dr. Jacqueline A Plumbridge, (genetics and molecular biology of E. coli);
- Dr. Eduardo Horjales, (cristallography and modelization);
PUBLICATIONS
1. Altamirano, M.M., Plumbridge, J.A., and Calcagno, M.L. (1993) Glucosamine-6-phosphate
deaminase from Escherichia coli has trimer of dimers structure with three
intersubunit disulfides. Biochem. J. 295: 645-648.
2. Altamirano, M.M., Hernández-Arana, A., Tello-Solís,
S., and Calcagno, M.L. (1994) Spectrochemical evidences for the presence
of a tyrosyl residue in the allosteric site of glucosamine-6-phosphate
deaminase from Escherichia coli. Eur. J. Biochem. 220: 409-413
3. Altamirano, M.M., Plumbridge, J.A., Horjales, E. and Calcagno, M.L
(1995). Asymmetric allosteric activation of Escherichia coli glucosamine
6-phosphate deaminase produced by replacements of Tyr 121. Biochemistry
34, 6074-6082
4. Oliva, G., Fontes, M.R.M., Garratt, R.C., Altamirano, M.M., Calcagno, M.L.
and Horjales, E. (1995) Structure and catalytic mechanism of glucosamine-6-phosphate
deaminase from Escherichia coli. at 2.1Å resolution. Structure
3: 1323-1332.
5. Souza, José M., Plumbridge, J.A. and Calcagno, M.L
N-Acetylglucosamine 6-phosphate deacetylase from Escherichia coli:
molecular and kinetic properties. Arch. Biochem. Biophys. (1997)
340 : 338-346.
6. Montero-Morán Gabriela M., Horjales, E, Calcagno M.L. and Altamirano
M.M.
Tyr254 hydroxyl group acts as a two-way switch mechanism in the coupling
of heterotropic and homotropic effects in Escherichia coli glucosamine-6-phosphate
deaminase. Biochemistry (1998) 37:7844-7849.
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