Structural analysis of Escherichia coli exopolyphosphatase
Abstract
A largely forgotten molecule, polyphosphate (poly P) has been found in every cell examined yet its function remains unclear. In E. coli, poly P is found to accumulate in response to phosphate and amino acid starvation, which results in degradation of free ribosomal proteins to supply amino acids for survival during nutrient poor conditions. When cells are no longer starved for amino acids, exopolyphosphatase (Ppx) hydrolyzes the accumulated poly P and terminates the degradation of ribosomal proteins. In E. coli, Ppx processively hydrolyzes the terminal phosphate from long poly P chains of approximately 500 to 700 phosphates until pyrophosphate is reached. Ppx has been predicted to be a member of the ASKHA (Acetate and Sugar Kinases, Hsp70, Actin) superfamily of phosphotransferases. The ASKHA superfamily shares a conserved core structure with the topology βββαβαβα, which is duplicated to make two domains. Ppx is a dimer of 58 kDa subunits and is made up of three domains. The first two domains are responsible for hydrolysis and the third domain binds poly P. We have solved the X-ray crystal structure of E. coli Ppx to 1.9Å resolution. The structure of Ppx confirms the prediction that it would have the same conserved core structure of the ASKHA superfamily. It has been determined that Domains I and II possess the conserved core structure. Domain III is made up of seven α-helices and a three-stranded β-sheet. Although the structure of Ppx has been determined without bound poly P, many sulfate ions are bound and are thought to represent phosphates in the poly P chain. Sulfate ions have been found in the active site, which lies between domains I and II, and are in contact with residues that are thought to be important for hydrolysis of the poly P chain. Domain III also has sulfate ions bound. These sulfates may give clues as to where poly P binds in this domain. Unexpectedly, the structure of Ppx has revealed a dimeric structure different to those ASKHA members that form dimers.
Degree
Ph.D.
Advisors
Sanders, Purdue University.
Subject Area
Biophysics|Biochemistry
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