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 up to several thousand phosphate residues in length is known to accumulate in bacteria, fungi, plants, and animals (1). The function of this phosphate biopolymer, although not well understood, is believed to involve energy and phosphate storage, transformation of DNA across cell membranes (2), gene regulation (3), stationary phase survival (4, 5), and response to starvation for amino acids or carbon (6, 7).Exopolyphosphatases are found in a variety of organisms and hydrolyze terminal phosphate bonds to yield orthophosphate (P 1 ). 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 P 40 for guanosine pentaphosphate phosphohydrolase (GppA) (8) to P 100 , roughly 200-Å in length, for polyphosphate glucokinase of Propionibacterium shermani (9, 10).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. EXPERIMENTAL PROCEDURESPPX Assays-Radioactive [ 32 P]polyphosphate was synthesized according to reported procedures (11,12). This polyphosphate was used to assay PPX activity with minor modifications to reported methods (8, 13). The 15-l reactions contained 50 mM Tricine-KOH (pH 8.0), 1 mM MgCl 2 , 175 mM KCl, and 100 nM [ 32 P]polyphosphate chains, assuming a chain length of 750 phosphate residue...

Bioremediation promises to be an important technique in the removal of trichloroethene (TCE) and tetrachloroethene (PCE) from contaminated waste sites and contaminated groundwater systems. However, the use of aerobic degradation to degrade these compounds is not always possible. Thus, anaerobic degradation is a promising alternative that may be used to remediate these sites. Recently, literature reports indicate complete anaerobic dechlorination of TCE and PCE by microorganisms enriched from wastewater treatment plants. We report here the complete dechlorination of TCE to ethene in anaerobic microcosms by microorganisms enriched from a TCE contaminated groundwater aquifer using glucose as an electron donor. Initial TCE degradation activity occurred after 10 days of incubation and TCE was no longer detected after 20 days of incubation. During the incubation period, the reductive dechlorination products associated with TCE degradation were detected. Ultimately, all of the TCE was converted to ethene. The glucose culture was further enriched and demonstrated increased rates of TCE conversion to ethene. Our results show that organisms isolated from a contaminated groundwater site are capable of completely degrading TCE to ethene at appreciable rates, and indicate the potential of using in situ anaerobic bioremediation to clean up TCE contaminated sites.

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