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J. Biol. Chem., Vol. 283, Issue 19, 12909-12918, May 9, 2008

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Enzymatic Properties of an Ecto-nucleoside Triphosphate Diphosphohydrolase from Legionella pneumophila

SUBSTRATE SPECIFICITY AND REQUIREMENT FOR VIRULENCE^*

Fiona M. Sansom¹, Patrice Riedmaier, Hayley J. Newton², Michelle A. Dunstone^¶3, Christa E. Müller^||, Holger Stephan^**, Emma Byres^¶, Travis Beddoe^¶, Jamie Rossjohn^¶, Peter J. Cowan, Anthony J. F. d'Apice, Simon C. Robson^¶¶, and Elizabeth L. Hartland⁴

From the Departments of Microbiology and Immunology and Medicine, University of Melbourne, Parkville, Victoria 3010, Australia, the Departments of Microbiology and ^¶Biochemistry and Molecular Biology, Monash University, Clayton, Victoria 3800, Australia, the ^||University of Bonn, Pharmacology Institute, Bonn D53121, Germany, the ^**Research Centre Dresden-Rossendorf, Institute of Radiopharmacy, D-01314 Dresden, Germany, the Immunology Research Centre, St. Vincent's Hospital, Fitzroy, Victoria 3065, Australia, and the ^¶¶Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts 02215

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.

Received for publication, February 7, 2008 , and in revised form, March 7, 2008.

^* This work was supported, in whole or in part, by National Institutes of Health Grants HL57307, HL63972, and HL076540 (to S. C. R.). This work was also supported by an Australian National Health and Medical Research Council grant (to E. L. H., A. J. F. d'A., and P. J. C.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ Recipient of a Monash graduate scholarship and a Monash University travel grant.

² Recipient of an Australian Postgraduate Award.

³ Recipient of an Australian National Health and Medical Research Council Peter Doherty fellowship.

⁴ To whom correspondence should be addressed: Dept. of Microbiology and Immunology, University of Melbourne, Parkville, Victoria 3010, Australia. Tel.: 61-3-83448041; Fax: 61-3-93471540; E-mail: hartland{at}unimelb.edu.au.

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				F. M. Sansom, S. C. Robson, and E. L. Hartland Possible Effects of Microbial Ecto-Nucleoside Triphosphate Diphosphohydrolases on Host-Pathogen Interactions Microbiol. Mol. Biol. Rev., December 1, 2008; 72(4): 765 - 781. [Abstract] [Full Text] [PDF]