title: Ala160 and His116 residues are involved in activity and specificity of apyrase, an ATP-hydrolysing enzyme produced by enteroinvasive Escherichia coli.
abstract: The virulence plasmid-carried apy (phoN2) gene of Shigella and related enteroinvasive Escherichia coli (EIEC) encodes apyrase, an ATP-diphosphohydrolase belonging to class A of the non-specific acid phosphatases (A-NSAPs). Apyrase and A-NSAPs share three domains of conserved amino acids (domains D1-D3) containing residues forming the putative active site of apyrase. In spite of their similarity, apyrase and A-NSAPs show different substrate specificity, apyrase being able to hydrolyse nucleotide tri- and diphosphates, but not monophosphates, as well as p-nitrophenyl phosphate (pNPP), while A-NSAPs are also active towards monophosphates and pNPP. In this paper, to get further insights into the structure-function relationship of apyrase, a random and site-directed mutagenesis of the apy gene of EIEC strain HN280 was conducted. Results indicate that amino acids located within the D2 and D3 conserved domains (Ser157 and Arg192, respectively) as well as residues located in the N-terminal (Ser97) and C-terminal (Glu233) domains are required for enzyme activity. Surprisingly, Ala160, located near the D2 domain and considered to be important for enzyme specificity, is required for enzyme activity, as its substitution with Thr led to the inactivation of enzyme activity. Furthermore, residue His116 is involved in apyrase specificity, since the H116L apyrase mutant shows substrate specificity resembling that of A-NSAPs.
authors.affiliation: Dipartimento di Scienze di Sanità Pubblica, Università di Roma 'La Sapienza', Piazzale A. Moro, 5 00185 Rome, Italy.
authors.forename: Serena
authors.initials: S
authors.lastname: Sarli
authors.affiliation: Dipartimento di Scienze Biomediche, Sezione di Microbiologia, Università G. D'Annunzio, Chieti, Italy.
authors.forename: Mauro
authors.initials: M
authors.lastname: Nicoletti
authors.affiliation: Dipartimento di Scienze di Sanità Pubblica, Università di Roma 'La Sapienza', Piazzale A. Moro, 5 00185 Rome, Italy.
authors.forename: Serena
authors.initials: S
authors.lastname: Schippa
authors.affiliation: Dipartimento di Scienze di Sanità Pubblica, Università di Roma 'La Sapienza', Piazzale A. Moro, 5 00185 Rome, Italy.
authors.forename: Federica
authors.initials: F
authors.lastname: Del Chierico
authors.affiliation: Dipartimento di Scienze Biomediche, Sezione di Microbiologia, Università G. D'Annunzio, Chieti, Italy.
authors.forename: Daniela
authors.initials: D
authors.lastname: Santapaola
authors.affiliation: Dipartimento di Medicina Sperimentale, Seconda Università, Naples, Italy.
authors.forename: Piera
authors.initials: P
authors.lastname: Valenti
authors.affiliation: Dipartimento di Scienze di Sanità Pubblica, Università di Roma 'La Sapienza', Piazzale A. Moro, 5 00185 Rome, Italy.
authors.forename: Francesca
authors.initials: F
authors.lastname: Berlutti
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism*; D000409:Alanine / Q000378:metabolism; D001081:Apyrase / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics / Q000472:pathogenicity; D015964:Gene Expression Regulation, Bacterial; D006639:Histidine / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D010957:Plasmids / Q000235:genetics*; D013379:Substrate Specificity; D037521:Virulence Factors
chemical_list: D037521:Virulence Factors; D006639:Histidine; D000255:Adenosine Triphosphate; D001081:Apyrase; D000409:Alanine