|Gene:||ppk||Accession Numbers: EG11510 (MetaCyc), b2501, ECK2497|
Species: Escherichia coli K-12 substr. MG1655
Component of: degradosome (extended summary available)
Subunit composition of polyphosphate kinase = [Ppk]2
Polyphosphate kinase (PPK) catalyses several reactions in E. coli K-12.
PPK transfers the γ phosphate of ATP processively to generate inorganic polyphosphate (polyP or polyPi) and ADP. Phosphorylated enzyme (PPK-P) is an intermediate in this reaction. Purified PPK is a tetramer and requires Mg2+ for activity [Ahn90]. PPK in which histidine 435 or histidine 454 have been altered to glutamine or alanine results in enzyme that is unable to autophosphorylate and lacks polyphosphate kinase activity. Purified PPK catalyses the synthesis of polyP chains with a uniform length of 750 +/- 50 phosphate groups. No intermediate chain lengths were visualised and polyP chains ranging from 2 - 40 residues in length failed to act as primers for the synthesis reaction in vitro [Kumble96].
PPK also catalyses the reverse reaction which synthesizes ATP from inorganic polyphosphate and ADP [Ahn90]. Partially purified PPK from E. coli B is most active in ATP synthesis using polyphosphate molecules with a chain length greater than 132 and its activity decreases with decreasing chain length [Haeusler92].
Purified PPK can transfer a phosphate from inorganic polyphosphate to nucleotide diphosphates including ADP, GDP, CDP, UDP, dADP, dGDP, dCDP and TDP. It can also transfer a pyrophosphate group to GDP to form guanosine 5' tetraphosphate (ppGpp) [Kuroda97a].
Overproduced PPK results in increased polyPi:AMP phosphotransferase (PAP) activity in E. coli K-12. PPK requires adenylate kinase (ADK) for PAP activity. PPK and ADK from a complex in the presence of polyPi in vitro [Ishige00].
Overproduced PPK is associated with the outer membrane [Akiyama92a].
Cells overexpressing PPK have increased polyphosphate levels (estimated at 220 µg/1011 cells) compared to wild type (2 µg/1011 cells) while ppk::kan cells have decreased polyphosphate levels (0.16 µg/1011 cells). ppk::kan cells are more sensitive to hydrogen peroxide stress and to heat stress and show reduced survival in stationary phase [Crooke94, Rao96]. PPK is required to stimulate protein degradation upon nutritional downshift [Kuroda99].
ppk forms an operon with ppx, encoding an exopolyphosphatase [Akiyama93]
PPK has been identified as a component of the E. coli RNA degradosome [Blum97].
Native PPK purifies as a tetramer however different subunit organizations may be associated with the different functions of the enzyme. Experiments using radiation inactivation of enzyme activity suggest that the functional unit for PPK activity (both forward and reverse) is a dimer, the functional unit for autophosphorylation is a tetramer (at 5mM ATP) or dimer (at 1mM ATP) and the functional unit for ppGpp synthesis is a trimer [Tzeng00]
Crystal structures have been determined for polyphosphate kinase on its own and binding the non-hydrolysable ATP analogue AMP-PNP. The structure is an interlocked dimer. Each monomer has 4 structural domains: an amino terminal or N domain; a head domain and two carboxy terminal domains C1 and C2. A tunnel structure penetrates the centre of each monomer and contains the sites of catalysis. Histidine residue 435 directly interacts with the AMP-PNP γ phosphate group and probably represents the site of autophosphorylation [Zhu03, Zhu05a].
Locations: outer membrane, inner membrane, cytosol
|Map Position: [2,621,066 -> 2,623,132]|
Molecular Weight of Polypeptide: 80.431 kD (from nucleotide sequence), 69.0 kD (experimental) [Ahn90 ]
Molecular Weight of Multimer: 270.0 kD (experimental) [Ahn90]
Unification Links: ASAP:ABE-0008235 , CGSC:32894 , DIP:DIP-36218N , EchoBASE:EB1472 , EcoGene:EG11510 , EcoliWiki:b2501 , Mint:MINT-1244108 , OU-Microarray:b2501 , PortEco:ppk , PR:PRO_000023580 , Pride:P0A7B1 , Protein Model Portal:P0A7B1 , RefSeq:NP_416996 , RegulonDB:EG11510 , SMR:P0A7B1 , String:511145.b2501 , UniProt:P0A7B1
Relationship Links: InterPro:IN-FAMILY:IPR001736 , InterPro:IN-FAMILY:IPR003414 , InterPro:IN-FAMILY:IPR024953 , InterPro:IN-FAMILY:IPR025198 , InterPro:IN-FAMILY:IPR025200 , PDB:Structure:1XDO , PDB:Structure:1XDP , Pfam:IN-FAMILY:PF02503 , Pfam:IN-FAMILY:PF13089 , Pfam:IN-FAMILY:PF13090 , Prosite:IN-FAMILY:PS50035
|Biological Process:||GO:0006757 - ADP phosphorylation
GO:0006799 - polyphosphate biosynthetic process [GOA06, GOA01, Ahn90]
GO:0046777 - protein autophosphorylation [Ishige00]
GO:0008152 - metabolic process [GOA01]
GO:0016310 - phosphorylation [UniProtGOA11]
|Molecular Function:||GO:0005515 - protein binding
GO:0008976 - polyphosphate kinase activity [GOA06, GOA01a, GOA01, Ahn90]
GO:0016776 - phosphotransferase activity, phosphate group as acceptor [Kuroda97a]
GO:0016778 - diphosphotransferase activity [Kuroda97a]
GO:0042803 - protein homodimerization activity [Zhu05a]
GO:0043751 - polyphosphate:AMP phosphotransferase activity [Ishige00]
GO:0000166 - nucleotide binding [UniProtGOA11]
GO:0003824 - catalytic activity [GOA01]
GO:0005524 - ATP binding [UniProtGOA11]
GO:0016301 - kinase activity [UniProtGOA11]
GO:0016740 - transferase activity [UniProtGOA11]
|Cellular Component:||GO:0005829 - cytosol
GO:0009358 - polyphosphate kinase complex [GOA01, Ahn90, Zhu05a]
GO:0031241 - periplasmic side of cell outer membrane [Akiyama92a]
GO:0005886 - plasma membrane [UniProtGOA11a, UniProtGOA11]
GO:0016020 - membrane [UniProtGOA11]
|MultiFun Terms:||metabolism → metabolism of other compounds → phosphorous metabolism|
Enzymatic reaction of: polyphosphate kinase
Synonyms: polyphosphate phosphotransferase
EC Number: 220.127.116.11
The reaction direction shown, that is, A + B ↔ C + D versus C + D ↔ A + B, is in accordance with the direction of enzyme catalysis.
This reaction is reversible. [Ahn90]
Subunit of: degradosome
Species: Escherichia coli K-12 substr. MG1655
Subunit composition of
degradosome = [(Ppk)2][(Rne)4][(RhlB)2][(Pnp)3][(Eno)2]
polyphosphate kinase = (Ppk)2 (extended summary available)
ribonuclease E = (Rne)4 (extended summary available)
RNase E = Rne
RhlB, ATP-dependent RNA helicase of the RNA degradosome = (RhlB)2 (extended summary available)
polynucleotide phosphorylase = (Pnp)3 (extended summary available)
polynucleotide phosphorylase monomer = Pnp
enolase = (Eno)2 (extended summary available)
The degradosome is a large, multiprotein complex involved in RNA degradation. It consists of the RNA degradation enzymes RNase E and PNPase, as well as the ATP-dependent RNA helicase RhlB and the metabolic enzyme enolase [Py94, Carpousis94, Py96]. Polyphosphate kinase and the chaperone protein DnaK are also associated with and may be components of the degradosome [Blum97, Miczak96]. A "minimal" degradosome composed of only RNase E, PNPase and RhlB degrades malEF REP RNA in an ATP-dependent manner in vitro, with activity equivalent to purified whole degradosomes. RNase E enzymatic function is dispensible for this test case, whereas PNPase must be catalytically active and incorporated into the degradosome for degradation to occur [Coburn99]. Based on immunogold labeling studies, RhlB and RNase E are present in equimolar quantities in the degradosome, which is tethered to the cytoplasmic membrane via the amino-terminus of RNase E [Liou01].
RNase E provides the organizational structure for the degradosome. Its carboxy-terminal half binds PNPase, RhlB and enolase, and the loss of this portion of the protein prevents degradation of a number of degradosome substrates, including the ptsG and mukB mRNAs and RNA I [Kido96, Vanzo98, Morita04]. This scaffold region is flexible, with isolated segments of increased structure that may be involved in binding other degradosome constituents [Callaghan04]. RNase E binding to partner proteins can be selectively disrupted. Loss of RhlB and enolase binding results in reduced degradosome activity. Conversely, disrupted PNPase binding yields increased activity. Strains any alteration in RNase E binding do not grow as well as wild type [Leroy02]. The amino-terminal half of RNase E contains sequences involved in oligomerization [Vanzo98].
In vitro purified degradosome generates 147-nucleotide RNase E cleavage intermediates from rpsT mRNA. Continuous cycles of polyadenylation and PNPase cleavage are necessary and sufficient to break down these intermediates, though RNase II can block this second degradation step [Coburn98]. RNAs with 3' REP stabilizers or stem loops must be polyadenylated to allow breakdown by the degradosome [Khemici04, Blum99]. Poly(G) and poly(U) tails do not allow degradation, though addition of a stretch of mixed nucleotides copied from within a coding region has stimulated degradation of a test substrate [Blum99].
The DEAD-box helicases SrmB, RhlE and CsdA bind RNase E in vitro at a different site than RhlB. RhlE and CsdA can both replace RhlB in promoting PNPase activity in vitro [Khemici04a]. CsdA is induced by cold shock, and following a shift to 15 degrees C it copurifies with the degradosome [PrudhommeGenere04].
At least two poly(A)-binding proteins interact with the degradosome. The cold-shock protein CspE inhibits internal cleavage and breakdown of polyadenylated RNA by RNase E and PNPase by blocking digestion through the poly(A) tail. S1, a component of the 30S ribosome, binds to RNase E and PNPase without apparent effect on their activities [Feng01].
The global effects of mutations in degradeosome constituents on mRNA levels have been evaluated using microarrays [Bernstein04].
Locations: inner membrane
|Cellular Component:||GO:0005886 - plasma membrane [Liou01]|
|Feature Class||Location||Common Name||Citations||Comment|
|Alpha-Helix-Region||2 -> 106||N domain|
|Chain||2 -> 688|
|Protein-Structure-Region||107 -> 321||H (head) domain|
|Protein-Structure-Region||322 -> 502||C1 domain|
|Conserved-Region||430 -> 464|
|Protein-Structure-Region||503 -> 687||C2 domain|
10/20/97 Gene b2501 from Blattner lab Genbank (v. M52) entry merged into EcoCyc gene EG11510; confirmed by SwissProt match.
Akiyama92a: Akiyama M, Crooke E, Kornberg A (1992). "The polyphosphate kinase gene of Escherichia coli. Isolation and sequence of the ppk gene and membrane location of the protein." J Biol Chem 1992;267(31);22556-61. PMID: 1331061
Bernstein04: Bernstein JA, Lin PH, Cohen SN, Lin-Chao S (2004). "Global analysis of Escherichia coli RNA degradosome function using DNA microarrays." Proc Natl Acad Sci U S A 101(9);2758-63. PMID: 14981237
Bessarab98: Bessarab DA, Kaberdin VR, Wei CL, Liou GG, Lin-Chao S (1998). "RNA components of Escherichia coli degradosome: evidence for rRNA decay." Proc Natl Acad Sci U S A 95(6);3157-61. PMID: 9501232
Blum99: Blum E, Carpousis AJ, Higgins CF (1999). "Polyadenylation promotes degradation of 3'-structured RNA by the Escherichia coli mRNA degradosome in vitro." J Biol Chem 274(7);4009-16. PMID: 9933592
Callaghan04: Callaghan AJ, Aurikko JP, Ilag LL, Gunter Grossmann J, Chandran V, Kuhnel K, Poljak L, Carpousis AJ, Robinson CV, Symmons MF, Luisi BF (2004). "Studies of the RNA degradosome-organizing domain of the Escherichia coli ribonuclease RNase E." J Mol Biol 340(5);965-79. PMID: 15236960
Carpousis94: Carpousis AJ, Van Houwe G, Ehretsmann C, Krisch HM (1994). "Copurification of E. coli RNAase E and PNPase: evidence for a specific association between two enzymes important in RNA processing and degradation." Cell 76(5);889-900. PMID: 7510217
Coburn99: Coburn GA, Miao X, Briant DJ, Mackie GA (1999). "Reconstitution of a minimal RNA degradosome demonstrates functional coordination between a 3' exonuclease and a DEAD-box RNA helicase." Genes Dev 13(19);2594-603. PMID: 10521403
Feng01: Feng Y, Huang H, Liao J, Cohen SN (2001). "Escherichia coli poly(A)-binding proteins that interact with components of degradosomes or impede RNA decay mediated by polynucleotide phosphorylase and RNase E." J Biol Chem 276(34);31651-6. PMID: 11390393
Haeusler92: Haeusler PA, Dieter L, Rittle KJ, Shepler LS, Paszkowski AL, Moe OA (1992). "Catalytic properties of Escherichia coli polyphosphate kinase: an enzyme for ATP regeneration." Biotechnol Appl Biochem 1992;15(2);125-33. PMID: 1316760
Hoffman88: Hoffman RC, Wyman PL, Smith LE, Nolt CL, Conley JL, Hevel JM, Warren JP, Reiner GA, Moe OA (1988). "Immobilized polyphosphate kinase: preparation, properties, and potential for use in adenosine 5'-triphosphate regeneration." Biotechnol Appl Biochem 10(2);107-17. PMID: 2838045
Ishige00: Ishige K, Noguchi T (2000). "Inorganic polyphosphate kinase and adenylate kinase participate in the polyphosphate:AMP phosphotransferase activity of Escherichia coli." Proc Natl Acad Sci U S A 2000;97(26);14168-71. PMID: 11106368
Khemici04: Khemici V, Carpousis AJ (2004). "The RNA degradosome and poly(A) polymerase of Escherichia coli are required in vivo for the degradation of small mRNA decay intermediates containing REP-stabilizers." Mol Microbiol 51(3);777-90. PMID: 14731278
Khemici04a: Khemici V, Toesca I, Poljak L, Vanzo NF, Carpousis AJ (2004). "The RNase E of Escherichia coli has at least two binding sites for DEAD-box RNA helicases: functional replacement of RhlB by RhlE." Mol Microbiol 54(5);1422-30. PMID: 15554979
Kido96: Kido M, Yamanaka K, Mitani T, Niki H, Ogura T, Hiraga S (1996). "RNase E polypeptides lacking a carboxyl-terminal half suppress a mukB mutation in Escherichia coli." J Bacteriol 178(13);3917-25. PMID: 8682798
Kuroda97a: Kuroda A, Kornberg A (1997). "Polyphosphate kinase as a nucleoside diphosphate kinase in Escherichia coli and Pseudomonas aeruginosa." Proc Natl Acad Sci U S A 1997;94(2);439-42. PMID: 9012801
Kuroda99: Kuroda A, Tanaka S, Ikeda T, Kato J, Takiguchi N, Ohtake H (1999). "Inorganic polyphosphate kinase is required to stimulate protein degradation and for adaptation to amino acid starvation in Escherichia coli." Proc Natl Acad Sci U S A 1999;96(25);14264-9. PMID: 10588694
Lasserre06: Lasserre JP, Beyne E, Pyndiah S, Lapaillerie D, Claverol S, Bonneu M (2006). "A complexomic study of Escherichia coli using two-dimensional blue native/SDS polyacrylamide gel electrophoresis." Electrophoresis 27(16);3306-21. PMID: 16858726
Leroy02: Leroy A, Vanzo NF, Sousa S, Dreyfus M, Carpousis AJ (2002). "Function in Escherichia coli of the non-catalytic part of RNase E: role in the degradation of ribosome-free mRNA." Mol Microbiol 45(5);1231-43. PMID: 12207692
LinChao99: Lin-Chao S, Wei CL, Lin YT (1999). "RNase E is required for the maturation of ssrA RNA and normal ssrA RNA peptide-tagging activity." Proc Natl Acad Sci U S A 96(22);12406-11. PMID: 10535935
Liou01: Liou GG, Jane WN, Cohen SN, Lin NS, Lin-Chao S (2001). "RNA degradosomes exist in vivo in Escherichia coli as multicomponent complexes associated with the cytoplasmic membrane via the N-terminal region of ribonuclease E." Proc Natl Acad Sci U S A 98(1);63-8. PMID: 11134527
Morita04: Morita T, Kawamoto H, Mizota T, Inada T, Aiba H (2004). "Enolase in the RNA degradosome plays a crucial role in the rapid decay of glucose transporter mRNA in the response to phosphosugar stress in Escherichia coli." Mol Microbiol 54(4);1063-75. PMID: 15522087
PrudhommeGenere04: Prud'homme-Genereux A, Beran RK, Iost I, Ramey CS, Mackie GA, Simons RW (2004). "Physical and functional interactions among RNase E, polynucleotide phosphorylase and the cold-shock protein, CsdA: evidence for a 'cold shock degradosome'." Mol Microbiol 54(5);1409-21. PMID: 15554978
Tzeng00: Tzeng CM, Kornberg A (2000). "The multiple activities of polyphosphate kinase of Escherichia coli and their subunit structure determined by radiation target analysis." J Biol Chem 2000;275(6);3977-83. PMID: 10660553
Van97: Van Dien SJ, Keyhani S, Yang C, Keasling JD (1997). "Manipulation of independent synthesis and degradation of polyphosphate in Escherichia coli for investigation of phosphate secretion from the cell." Appl Environ Microbiol 1997;63(5);1689-95. PMID: 9143103
Vanzo98: Vanzo NF, Li YS, Py B, Blum E, Higgins CF, Raynal LC, Krisch HM, Carpousis AJ (1998). "Ribonuclease E organizes the protein interactions in the Escherichia coli RNA degradosome." Genes Dev 12(17);2770-81. PMID: 9732274
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