Abstract
Triphosphate tunnel metalloenzymes (TTMs) are a newly recognized superfamily of phosphotransferases defined by a unique active site residing within an eight-stranded beta barrel. The prototypical members are the eukaryal metal-dependent RNA triphosphatases, which catalyze the initial step in mRNA capping. Little is known about the activities and substrate specificities of the scores of TTM homologs present in bacterial and archaeal proteomes, nearly all of which are annotated as adenylate cyclases. Here we have conducted a biochemical and structure-function analysis of a TTM protein (CthTTM) from the bacterium Clostridium thermocellum. CthTTM is a metal-dependent tripolyphosphatase and nucleoside triphosphatase; it is not an adenylate cyclase. We have identified 11 conserved amino acids in the tunnel that are critical for tripolyphosphatase and ATPase activity. The most salient findings are that (i) CthTTM is 150-fold more active in cleaving tripolyphosphate than ATP and (ii) the substrate specificity of CthTTM can be transformed by a single mutation (K8A) that abolishes tripolyphosphatase activity while strongly stimulating ATP hydrolysis. Our results underscore the plasticity of CthTTM substrate choice and suggest how novel specificities within the TTM superfamily might evolve through changes in the residues that line the tunnel walls.
Highlights
Cal property of this branch of the tunnel metalloenzymes (TTMs) superfamily is the ability to hydrolyze NTPs to nucleoside diphosphates and Pi in the presence of manganese [2]
The NeuTTM fold is distinguished by the fact that the eight-stranded antiparallel  barrel is not a closed tunnel but rather a C-shaped “cup” that is prevented from closing by virtue of the insertion of a broken C-terminal helix into one end of the tunnel (Fig. 1B)
The rate of release of 32Pi from [␥-32P]ATP was identical to the rate of conversion of [␣-32P]ATP to [␣-32P]ADP in parallel reaction mixtures containing the same enzyme concentrations (Fig. 2B), indicating that CthTTM catalyzed the hydrolysis of ATP to ADP and Pi
Summary
Cal property of this branch of the TTM superfamily is the ability to hydrolyze NTPs to nucleoside diphosphates and Pi in the presence of manganese [2]. Tripolyphosphatase Assay—Reaction mixtures (50 l) containing 50 mM Tris-HCl, pH 9.0, 10 mM MgCl2 or 0.5 mM MnCl2, 100 M inorganic tripolyphosphate (PPPi) (Sigma), and CthTTM, as specified, were incubated for 30 min at 37 °C. The ATP and tripolyphosphatase activity profiles peaked in fractions 20 –22 and coincided with an abundance of the CthTTM polypeptide (Fig. 5).
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