Engineering human Fhit, a diadenosine triphosphate hydrolase, into an efficient dinucleoside polyphosphate synthase.
The putative human tumor suppressor gene FHIT encodes Fhit, the fragile histidine triad protein. Fhit is thought to participate in a signal transduction pathway involving dinucleoside polyphosphates. Fhit catalyzes the Mg2+-dependent hydrolysis of P1-5'-O-adenosine-P3-5'-O-adenosine triphosphate (Ap3A) to AMP and MgADP. Mutation of His96 to glycine disables Fhit as a catalyst for the hydrolysis of phosphoanhydrides such as Ap3A. However, the mutated enzyme H96G-Fhit efficiently catalyzes the synthesis of phosphoanhydride bonds in reactions of nucleoside-5'-phosphimidazolides with nucleoside di- and triphosphates. H96G-Fhit can be employed in the synthesis of a wide range of dinucleoside tri- and tetraphosphates. We here describe the use of H96G-Fhit to catalyze the synthesis of Ap3A, Ap3C, Ap3G, Ap3T, Ap3U, Cp3U, Tp3U, dAp3U, Ap4A, Ap4U, and the fluorescent Ap4etheno-C.
Huang
Kaisheng
K
Department of Biochemistry, University of Wisconsin-Madison,1710 University Avenue, Madison, Wisconsin 53726, USA.
Frey
Perry A
PA
D017766:Acid Anhydride Hydrolases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D002384:Catalysis; D015226:Dinucleoside Phosphates / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D009363:Neoplasm Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D017856:Phosphotransferases (Phosphate Group Acceptor) / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D015202:Protein Engineering / Q000379:methods; D000099170:Bis(5'-adenosyl)triphosphatase
D017766:Acid Anhydride Hydrolases; D015226:Dinucleoside Phosphates; D009363:Neoplasm Proteins; D017856:Phosphotransferases (Phosphate Group Acceptor); D000099170:Bis(5'-adenosyl)triphosphatase; C011440:diadenosine triphosphate; C022424:polyphosphate kinase