title: A specific inorganic triphosphatase from Nitrosomonas europaea: structure and catalytic mechanism.
abstract: The CYTH superfamily of proteins is named after its two founding members, the CyaB adenylyl cyclase from Aeromonas hydrophila and the human 25-kDa thiamine triphosphatase. Because these proteins often form a closed β-barrel, they are also referred to as triphosphate tunnel metalloenzymes (TTM). Functionally, they are characterized by their ability to bind triphosphorylated substrates and divalent metal ions. These proteins exist in most organisms and catalyze different reactions depending on their origin. Here we investigate structural and catalytic properties of the recombinant TTM protein from Nitrosomonas europaea (NeuTTM), a 19-kDa protein. Crystallographic data show that it crystallizes as a dimer and that, in contrast to other TTM proteins, it has an open β-barrel structure. We demonstrate that NeuTTM is a highly specific inorganic triphosphatase, hydrolyzing tripolyphosphate (PPP(i)) with high catalytic efficiency in the presence of Mg(2+). These data are supported by native mass spectrometry analysis showing that the enzyme binds PPP(i) (and Mg-PPP(i)) with high affinity (K(d) < 1.5 μm), whereas it has a low affinity for ATP or thiamine triphosphate. In contrast to Aeromonas and Yersinia CyaB proteins, NeuTTM has no adenylyl cyclase activity, but it shares several properties with other enzymes of the CYTH superfamily, e.g. heat stability, alkaline pH optimum, and inhibition by Ca(2+) and Zn(2+) ions. We suggest a catalytic mechanism involving a catalytic dyad formed by Lys-52 and Tyr-28. The present data provide the first characterization of a new type of phosphohydrolase (unrelated to pyrophosphatases or exopolyphosphatases), able to hydrolyze inorganic triphosphate with high specificity.
authors.affiliation: GIGA-Neuroscience, University of Liège, B-4000 Liège, Belgium.
authors.forename: David
authors.initials: D
authors.lastname: Delvaux
authors.forename: Mamidanna R V S
authors.initials: MR
authors.lastname: Murty
authors.forename: Valérie
authors.initials: V
authors.lastname: Gabelica
authors.forename: Bernard
authors.initials: B
authors.lastname: Lakaye
authors.forename: Vladimir V
authors.initials: VV
authors.lastname: Lunin
authors.forename: Tatiana
authors.initials: T
authors.lastname: Skarina
authors.forename: Olena
authors.initials: O
authors.lastname: Onopriyenko
authors.forename: Gregory
authors.initials: G
authors.lastname: Kohn
authors.forename: Pierre
authors.initials: P
authors.lastname: Wins
authors.forename: Edwin
authors.initials: E
authors.lastname: De Pauw
authors.forename: Lucien
authors.initials: L
authors.lastname: Bettendorff
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics; D002384:Catalysis; D006867:Hydrolases / Q000737:chemistry* / Q000235:genetics; D008667:Metalloproteins / Q000737:chemistry* / Q000235:genetics; D043370:Nitrosomonas europaea / Q000201:enzymology* / Q000235:genetics; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D013329:Structure-Activity Relationship
chemical_list: D001426:Bacterial Proteins; D008667:Metalloproteins; D011994:Recombinant Proteins; D006867:Hydrolases