[en] 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 (PPPi) with high catalytic efficiency in the presence of Mg2+. These data are supported by native mass spectrometry analysis showing that the enzyme binds PPPi (and Mg-PPPi) with high affinity (Kd < 1.5 μM), while 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 Ca2+ and Zn2+ ions. We suggest a catalytic mechanism involving a catalytic dyad formed by K52 and Y28. 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.
Research Center/Unit :
Giga-Neurosciences - ULiège Giga-Systems Biology and Chemical Biology - ULiège Banting and Best Department of Medical Research, University of Toronto
Disciplines :
Biochemistry, biophysics & molecular biology
Author, co-author :
Delvaux, David ; Université de Liège - ULiège > Département des sciences biomédicales et précliniques > Biochimie et physiologie humaine et pathologique
Murty, Mamidana R.V.S
Gabelica, Valérie ; Université de Liège - ULiège > Département de chimie (sciences) > GIGA-R : Laboratoire de spectrométrie de masse (L.S.M.)
Lakaye, Bernard ; Université de Liège - ULiège > Département des sciences biomédicales et précliniques > Biochimie et physiologie humaine et pathologique
De Pauw, Edwin ; Université de Liège - ULiège > Département de chimie (sciences) > GIGA-R : Laboratoire de spectrométrie de masse (L.S.M.)
Bettendorff, Lucien ; Université de Liège - ULiège > Département des sciences biomédicales et précliniques > Biochimie et physiologie humaine et pathologique
Language :
English
Title :
A specific inorganic triphosphatase from Nitrosomonas europaea: structure and catalytic mechanism
Publication date :
2011
Journal title :
Journal of Biological Chemistry
ISSN :
0021-9258
eISSN :
1083-351X
Publisher :
American Society for Biochemistry and Molecular Biology, Baltimore, United States - Maryland
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