title: Enzymatic properties of an ecto-nucleoside triphosphate diphosphohydrolase from Legionella pneumophila: substrate specificity and requirement for virulence.
abstract: Legionella pneumophila is the predominant cause of Legionnaires disease, a severe and potentially fatal form of pneumonia. Recently, we identified an ecto-nucleoside triphosphate diphosphohydrolase (NTPDase) from L. pneumophila, termed Lpg1905, which enhances intracellular replication of L. pneumophila in eukaryotic cells. Lpg1905 is the first prokaryotic member of the CD39/NTPDase1 family of enzymes, which are characterized by the presence of five apyrase conserved regions and the ability to hydrolyze nucleoside tri- and diphosphates. Here we examined the substrate specificity of Lpg1905 and showed that apart from ATP and ADP, the enzyme catalyzed the hydrolysis of GTP and GDP but had limited activity against CTP, CDP, UTP, and UDP. Based on amino acid residues conserved in the apyrase conserved regions of eukaryotic NTPDases, we generated five site-directed mutants, Lpg1905E159A, R122A, N168A, Q193A, and W384A. Although the mutations E159A, R122A, Q193A, and W384A abrogated activity completely, N168A resulted in decreased activity caused by reduced affinity for nucleotides. When introduced into the lpg1905 mutant strain of L. pneumophila, only N168A partially restored the ability of L. pneumophila to replicate in THP-1 macrophages. Following intratracheal inoculation of A/J mice, none of the Lpg1905 mutants was able to restore virulence to an lpg1905 mutant during lung infection, thereby demonstrating the importance of NTPDase activity to L. pneumophila infection. Overall, the kinetic studies undertaken here demonstrated important differences to mammalian NTPDases and different sensitivities to NTPDase inhibitors that may reflect underlying structural variations.
authors.affiliation: Department of Microbiology and Immunology and Medicine, University of Melbourne, Parkville, Victoria 3010, Australia.
authors.forename: Fiona M
authors.initials: FM
authors.lastname: Sansom
authors.forename: Patrice
authors.initials: P
authors.lastname: Riedmaier
authors.forename: Hayley J
authors.initials: HJ
authors.lastname: Newton
authors.forename: Michelle A
authors.initials: MA
authors.lastname: Dunstone
authors.forename: Christa E
authors.initials: CE
authors.lastname: Müller
authors.forename: Holger
authors.initials: H
authors.lastname: Stephan
authors.forename: Emma
authors.initials: E
authors.lastname: Byres
authors.forename: Travis
authors.initials: T
authors.lastname: Beddoe
authors.forename: Jamie
authors.initials: J
authors.lastname: Rossjohn
authors.forename: Peter J
authors.initials: PJ
authors.lastname: Cowan
authors.forename: Anthony J F
authors.initials: AJ
authors.lastname: d'Apice
authors.forename: Simon C
authors.initials: SC
authors.lastname: Robson
authors.forename: Elizabeth L
authors.initials: EL
authors.lastname: Hartland
mesh_terms: D000244:Adenosine Diphosphate / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D001081:Apyrase / Q000235:genetics / Q000378:metabolism*; D002384:Catalysis; D002460:Cell Line; D003562:Cytidine / Q000378:metabolism; D004789:Enzyme Activation; D006153:Guanosine Diphosphate / Q000378:metabolism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis / Q000187:drug effects; D016952:Legionella pneumophila / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development / Q000472:pathogenicity*; D008670:Metals / Q000494:pharmacology; D051379:Mice; D009097:Multienzyme Complexes / Q000378:metabolism; D009154:Mutation / Q000235:genetics; D013379:Substrate Specificity; D014529:Uridine / Q000378:metabolism
chemical_list: D008670:Metals; D009097:Multienzyme Complexes; D006153:Guanosine Diphosphate; D003562:Cytidine; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D001081:Apyrase; D014529:Uridine