Mechanistic studies of Mycobacteriophage TM4 LysA combining Bioinformatic and Expermental approach

MARTÍN MARIANO; URDÁNIZ ESTEFANIA; DUMAS MARÍA VICTORIA; PIURI MARIANA; MARTÍ MARCELO

Rosario-Sta. Fé

Congreso; L Reunión anual de la Sociedad Argentina de investigaciones en Bioquímica y Biología molecular (SAIB); 2014

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 (LysA; 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 amino acid 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 (H226 and H335) and an aspartic acid (D347) as the possible Zn coordination residues. Optimized structures of the LysA active site model shows that Zn could have tetra, penta or hexa coordination states, being the tetra and penta the most commonly found. Also, the 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 the enzymatic activity was tested by a chloroform assay and by zymogram using M. luteus peptidoglycan as substrate.