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:

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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