Novel polyphosphate: amp phosphotransferase

Abstract

This invention relates to a novel polyphosphate: AMP phosphotransferase (PAP), a gene coding this PAP, and their use. The PAP has the following properties: (A) action: catalyzing of the following two reactions: NMP+PolyP (n) →NDP+PolyP (n-1) dNMP+PolyP (n) →dNDP+PolyP (n-1) (wherein NMP represents nucleoside monophosphate, NDP represents nucleoside diphosphate, dNMP represents deoxynucleoside monophosphate, dNDP represents deoxynucleoside diphosphate, n represents degree of polymerization of the polyphosphate which is an integer of up to 100); (B) substrate specificity: specific to AMP, GMP, IMP, dAMP, and dGMP, also acting with CMP, UMP, dCMP, and TMP; (C) molecular weight: about 55 to 56 Kd (kilodalton); and (D) specific activity: at least 70 units per 1 mg of enzyme protein,

1 - 9 . (canceled)  
     
     
         10 . A method for producing ATP, comprising contacting AMP with the polyphosphate: AMP phosphotransferase and adenylate kinase in the presence of polyphosphate as a phosphate donor to produce ATP, wherein the polyphosphate: AMP phosphotransferase comprises SEQ ID NO:1, is encoded by a polynucleotide that is 95% identical to SEQ ID NO:2, or is encoded by a polynucleotide that hybridizes under stringent conditions to SEQ ID NO: 2, wherein the stringent conditions comprise hybridizing in a solution of 5×SSC, 0.1% w/v N-layorylsarcosine sodium salt, 0.02% w/v sodium dodecyl sulfate, 0.5% w/v blocking agent for 20 hours at a temperature of 60° C.  
     
     
         11 . An ATP generation/regeneration system comprising AMP, polyphosphate, polyphosphate: AMP phosphotransferase, and adenylate kinase, wherein the polyphosphate: AMP phosphotransferase comprises SEQ ID NO:1, is encoded by a polynucleotide that is 95% identical to SEQ ID NO:2, or is encoded by a polynucleotide that hybridizes under stringent conditions to SEQ ID NO: 2, wherein the stringent conditions comprise hybridizing in a solution of 5×SSC, 0.1% w/v N-layorylsarcosine sodium salt, 0.02% w/v sodium dodecyl sulfate, 0.5% w/v blocking agent for 20 hours at a temperature of 60° C.  
     
     
         12 . A method of producing a compound with an ATP consuming enzymatic reaction, comprising regenerating ATP from AMP with a system comprising polyphosphate, polyphosphate: AMP phosphotransferase, and adenylate kinase, wherein the polyphosphate: AMP phosphotransferase comprises SEQ ID NO:1, is encoded by a polynucleotide that is 95% identical to SEQ ID NO:2, or is encoded by a polynucleotide that hybridizes under stringent conditions to SEQ ID NO: 2, wherein the stringent conditions comprise hybridizing in a solution of 5×SSC, 0.1% w/v N-layorylsarcosine sodium salt, 0.02% w/v sodium dodecyl sulfate, 0.5% w/v blocking agent for 20 hours at a temperature of 60° C.