Gp29 LysA of mycobacteriophage TM4 can hydrolyze peptidoglycan through an N-acetyl-muramoyl-L-alanine amidase activity.

Estefanía Urdániz, Mariano Martín, Florencia Payaslián, Lucas Alfredo Defelipe, Martín Dodes, Mariano Martinez, Pedro M Alzari, Gabriela Cabrera, Marcelo Adrián Martí, Mariana Piuri

Journal: Biochimica et biophysica acta. Proteins and proteomics 2022;1870(2):140745

PMID: 34906734

Full Text on Elsevier Science View on PubMed

Abstract

Bacteriophage endolysins are crucial for progeny release at the end of the lytic cycle. Mycobacteriophage's genomes carry a lysin A essential gene, whose product cleaves the peptidoglycan (PG) layer and a lysin B, coding for an esterase, that cleaves the linkage between the mycolic acids and the arabinogalactan-PG complex. Lysin A mycobacteriophage proteins are highly modular and in gp29 (LysA) of phage TM4 three distinctive domains were identified. By bioinformatics analysis the central module was previously found to be similar to an amidase-2 domain family with an N-acetylmuramoyl -L-alanine amidase activity. We demonstrated experimentally that purified LysA is able to lyse a suspension of Micrococcus lysodeikticus and can promote cell lysis when expressed in E. coli and Mycobacterium smegmatis. After incubation of LysA with MDP (Muramyl dipeptide, N-acetyl-muramyl-L-alanyl-D-isoglutamine) we detected the presence of N-acetylmuramic acid (NAcMur) and L-Ala- D- isoGlutamine (L-Ala-D-isoGln) corroborating the proposed muramidase activity of this enzyme. This protein was stabilized at acidic pH in the presence of Zn consistent with the increase of the enzymatic activity under these conditions. By homology modeling, we predicted that the Zn ion is coordinated by His 226, His 335, and Asp 347 and we also identified the amino acid Glu 290 as the catalytic residue. LysA activity was completely abolished in derived mutants on these key residues, suggesting that the PG hydrolysis solely relies on the central domain of the protein.

Address: Departamento de Química Biológica, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Buenos Aires, Argentina; Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Consejo Nacional de Investigaciones Científicas y Técnicas, Buenos Aires, Argentina.; Departamento de Química Biológica, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Buenos Aires, Argentina; Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Consejo Nacional de Investigaciones Científicas y Técnicas, Buenos Aires, Argentina; Departamento de Bioquímica Clínica, Facultad de Ciencias Químicas, Universidad Nacional de Córdoba, Córdoba, Argentina; Centro de Investigaciones en Bioquímica Clínica e Inmunología Consejo Nacional de Investigaciones Científicas y Técnicas (CIBICI-CONICET), Córdoba, Argentina.; Departamento de Química Biológica, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Buenos Aires, Argentina; Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Consejo Nacional de Investigaciones Científicas y Técnicas, Buenos Aires, Argentina; European Molecular Biology Laboratory - Hamburg Unit, Notkestrasse 85, Hamburg 22607, Germany.; Institut Pasteur, CNRS, Université de Paris, Unité de Microbiologie Structurale, F-75015 Paris, France.; Facultad de Ciencias Exactas y Naturales, Departamento de Química Orgánica, Universidad de Buenos Aires, Buenos Aires, Argentina; CONICET-Universidad de Buenos Aires, Unidad de Microanálisis y Métodos Físicos aplicados a la Química Orgánica (UMYMFOR), Buenos Aires, Argentina.; Departamento de Química Biológica, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Buenos Aires, Argentina; Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Consejo Nacional de Investigaciones Científicas y Técnicas, Buenos Aires, Argentina. Electronic address: [email protected].

DOI: 10.1016/j.bbapap.2021.140745

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MeSH Terms: Computational Biology, Endopeptidases, Escherichia coli, Galactans, Hydrolysis, Mass Spectrometry, Micrococcus, Muramic Acids, Mycobacteriophages, Mycobacterium smegmatis, N-Acetylmuramoyl-L-alanine Amidase, Peptidoglycan, Viral Proteins