IQUIBICEN   23947
INSTITUTO DE QUIMICA BIOLOGICA DE LA FACULTAD DE CIENCIAS EXACTAS Y NATURALES
Unidad Ejecutora - UE
congresos y reuniones científicas
Título:
Mechanistic studies of Mycobacteriophage TM4 LysA combining Bioinformatic and experimental approaches
Autor/es:
MARIANO MARTIN; ESTEFANIA URDANIZ; DUMAS MV; MARIANA PIURI; MARCELO MARTÍ
Lugar:
Rosario
Reunión:
Congreso; L Reunión Anual SAIB; 2014
Institución organizadora:
SAIB
Resumen:
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Bacteriophages are
widely used as biotechnology tools. Phage encoded lysins, proteins that lyse
bacterial cell walls, represent desirable alternatives as antimicrobial agents.
Mycobacteriophage TM4 hosts a lysin A (gp29). LysA
sequence was analyzed using the BLAST search engine and Pfam database. We
were able to identify a peptidoglycan-recognition domain (cd06583) of 125
aminoacid length (position 225 to 350) and a second domain belonging to Amidase
2 family (pfam01510) harboring the substrate binding and Zn hosting catalytic
sites. To unravel the reaction mechanism of LysA, we performed structural
bioinformatic calculations and in-vitro
experiments. Sequence analysis revealed two Histidines (positions H226 and H335)
and an Aspartic acid (D347) as the possible Zn coordination residues. Optimized
structures of the LysA active site model shows that Zinc could have tetra,
penta or hexa coordination states, being the tetra and penta the most commonly
found. Also, the presence of Glutamic acid (E290) is predicted to be a key
residue for the catalytic activity, acting as base. The expression profile of
the heterologous protein in E.coli
was evaluated and activity of the enzyme was tested by a chloroform- assay and
by zymogram using M. luteus
peptidoglycan as substrate.