Polyphosphate Binding and Chain Length Recognition ofEscherichia coli Exopolyphosphatase

Exopolyphosphatase of Escherichia coli (PPX) is a highly processive enzyme demonstrating the ability to recognize polyphosphates of specific lengths. The mechanisms responsible for the processivity and polymer length recognition of the enzyme were investigated in relation to the manner in which polyphosphate is bound to the enzyme. Multiple polyphosphate binding sites were identified on distant portions of the enzyme and were determined to be responsible for the polymer length recognition of the enzyme. In addition, two independently folded domains were identified. The N-terminal domain contained a quasi-processive polyphosphatase active site belonging to the sugar kinase/actin/hsp70 superfamily. The C-terminal domain contained a single polyphosphate binding site and was responsible for nearly all of the PPX affinity for polyphosphate. This domain was also found to confer a highly processive mode of action to PPX. Collectively, these results were used to describe the interaction of polyphosphate with PPX. Exopolyphosphatase of Escherichia coli (PPX) is a highly processive enzyme demonstrating the ability to recognize polyphosphates of specific lengths. The mechanisms responsible for the processivity and polymer length recognition of the enzyme were investigated in relation to the manner in which polyphosphate is bound to the enzyme. Multiple polyphosphate binding sites were identified on distant portions of the enzyme and were determined to be responsible for the polymer length recognition of the enzyme. In addition, two independently folded domains were identified. The N-terminal domain contained a quasi-processive polyphosphatase active site belonging to the sugar kinase/actin/hsp70 superfamily. The C-terminal domain contained a single polyphosphate binding site and was responsible for nearly all of the PPX affinity for polyphosphate. This domain was also found to confer a highly processive mode of action to PPX. Collectively, these results were used to describe the interaction of polyphosphate with PPX. polyphosphate of n phosphate residues exopolyphosphatase of E. coli N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine polymerase chain reaction Polyphosphate up to several thousand phosphate residues in length is known to accumulate in bacteria, fungi, plants, and animals (1Kulaev I.S. The Biochemistry of Inorganic Polyphosphates. John Wiley & Sons, Inc., New York1979Google Scholar). The function of this phosphate biopolymer, although not well understood, is believed to involve energy and phosphate storage, transformation of DNA across cell membranes (2Reusch R.N. Sadoff H.L. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 4176-4180Crossref PubMed Scopus (211) Google Scholar), gene regulation (3Kornberg A. J. Bacteriol. 1995; 1773: 491-496Crossref Google Scholar), stationary phase survival (4Rao N.N. Kornberg A. J. Bacteriol. 1996; 178: 1394-1400Crossref PubMed Google Scholar, 5Crooke E. Akiyama M. Rao N.N. Kornberg A. J. Biol. Chem. 1994; 269: 6290-6295Abstract Full Text PDF PubMed Google Scholar), and response to starvation for amino acids or carbon (6Cashel M. Rudd K.E. Neidhardt F.C. Escherichia coli and Salmonella typhimurium: Cellular and Molecular Biology. American Society for Microbiology, Washington, D. C.1987: 1410-1438Google Scholar, 7Spria B. Silberstein N. Yagil E. J. Bacteriol. 1995; 177: 4053-4058Crossref PubMed Google Scholar).Exopolyphosphatases are found in a variety of organisms and hydrolyze terminal phosphate bonds to yield orthophosphate (P1).1 Their metabolic role, however, is unclear, as they appear to waste the stored energy of the phosphate bonds.Escherichia coli exopolyphosphatase, like most exopolyphosphatases, is highly processive, as it hydrolyzes entire polyphosphate chains greater than 1000 phosphate residues in length to orthophosphate without release of polyphosphate intermediates.In contrast, several polyphosphate-degrading enzymes produce specific chain length intermediates by processively removing terminal phosphates from long chain polyphosphates until the specific length intermediate remains and is released. These intermediates range from P40for guanosine pentaphosphate phosphohydrolase (GppA) (8Keasling J.D. Bertsch L. Kornberg A. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 7029-7033Crossref PubMed Scopus (118) Google Scholar) to P100, roughly 200-Å in length, for polyphosphate glucokinase of Propionibacterium shermani (9Pepin C.A. Wood H.G. J. Biol. Chem. 1986; 261: 4476-4480Abstract Full Text PDF PubMed Google Scholar, 10Pepin C.A. Wood H.G. J. Biol. Chem. 1987; 262: 5223-5226Abstract Full Text PDF PubMed Google Scholar).We have investigated this release of specific polyphosphate intermediates by PPX under conditions non-optimal for activity. Furthermore, we have identified domains of the enzyme and their functions in polyphosphate binding and processivity in order to describe the interaction of polyphosphate with PPX. Polyphosphate up to several thousand phosphate residues in length is known to accumulate in bacteria, fungi, plants, and animals (1Kulaev I.S. The Biochemistry of Inorganic Polyphosphates. John Wiley & Sons, Inc., New York1979Google Scholar). The function of this phosphate biopolymer, although not well understood, is believed to involve energy and phosphate storage, transformation of DNA across cell membranes (2Reusch R.N. Sadoff H.L. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 4176-4180Crossref PubMed Scopus (211) Google Scholar), gene regulation (3Kornberg A. J. Bacteriol. 1995; 1773: 491-496Crossref Google Scholar), stationary phase survival (4Rao N.N. Kornberg A. J. Bacteriol. 1996; 178: 1394-1400Crossref PubMed Google Scholar, 5Crooke E. Akiyama M. Rao N.N. Kornberg A. J. Biol. Chem. 1994; 269: 6290-6295Abstract Full Text PDF PubMed Google Scholar), and response to starvation for amino acids or carbon (6Cashel M. Rudd K.E. Neidhardt F.C. Escherichia coli and Salmonella typhimurium: Cellular and Molecular Biology. American Society for Microbiology, Washington, D. C.1987: 1410-1438Google Scholar, 7Spria B. Silberstein N. Yagil E. J. Bacteriol. 1995; 177: 4053-4058Crossref PubMed Google Scholar). Exopolyphosphatases are found in a variety of organisms and hydrolyze terminal phosphate bonds to yield orthophosphate (P1).1 Their metabolic role, however, is unclear, as they appear to waste the stored energy of the phosphate bonds. Escherichia coli exopolyphosphatase, like most exopolyphosphatases, is highly processive, as it hydrolyzes entire polyphosphate chains greater than 1000 phosphate residues in length to orthophosphate without release of polyphosphate intermediates. In contrast, several polyphosphate-degrading enzymes produce specific chain length intermediates by processively removing terminal phosphates from long chain polyphosphates until the specific length intermediate remains and is released. These intermediates range from P40for guanosine pentaphosphate phosphohydrolase (GppA) (8Keasling J.D. Bertsch L. Kornberg A. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 7029-7033Crossref PubMed Scopus (118) Google Scholar) to P100, roughly 200-Å in length, for polyphosphate glucokinase of Propionibacterium shermani (9Pepin C.A. Wood H.G. J. Biol. Chem. 1986; 261: 4476-4480Abstract Full Text PDF PubMed Google Scholar, 10Pepin C.A. Wood H.G. J. Biol. Chem. 1987; 262: 5223-5226Abstract Full Text PDF PubMed Google Scholar). We have investigated this release of specific polyphosphate intermediates by PPX under conditions non-optimal for activity. Furthermore, we have identified domains of the enzyme and their functions in polyphosphate binding and processivity in order to describe the interaction of polyphosphate with PPX. We thank Arthur Kornberg (Stanford University, Stanford, CA) for providing the native ppx gene contained in plasmid pBC9 and Harvey W. Blanch (University of California, Berkeley) for technical insight and advice on this project.