title: Novel triphosphate phosphohydrolase activity of Clostridium thermocellum TTM, a member of the triphosphate tunnel metalloenzyme superfamily.
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.
authors.affiliation: Molecular Biology Program, Sloan-Kettering Institute, New York, New York 10021, USA.
authors.forename: Niroshika
authors.initials: N
authors.lastname: Keppetipola
authors.forename: Ruchi
authors.initials: R
authors.lastname: Jain
authors.forename: Stewart
authors.initials: S
authors.lastname: Shuman
mesh_terms: D000255:Adenosine Triphosphate / Q000737:chemistry; D000409:Alanine / Q000737:chemistry; D000595:Amino Acid Sequence; D001665:Binding Sites; D048013:Acetivibrio thermocellus / Q000201:enzymology*; D006868:Hydrolysis; D008670:Metals / Q000737:chemistry; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D043583:Nucleoside-Triphosphatase / Q000737:chemistry / Q000502:physiology*; D010744:Phosphoric Monoester Hydrolases / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D013997:Time Factors
chemical_list: D008670:Metals; D011994:Recombinant Proteins; D000255:Adenosine Triphosphate; D010744:Phosphoric Monoester Hydrolases; D043583:Nucleoside-Triphosphatase; D000409:Alanine