Accession Number:

ADA459340

Title:

Structural Analysis and Bioengineering of Thermostable Pyrococcus Furiosus Prolidase for Optimization of Organophosphorus Nerve Agent Detoxification

Descriptive Note:

Final rept. 22 Jul 2002-30 Jun 2006

Corporate Author:

NORTH CAROLINA STATE UNIV AT RALEIGH OFFICE OF CONTRACTS AND GRANTS

Report Date:

2006-06-01

Pagination or Media Count:

34.0

Abstract:

prolidase, aminopeptidase, dinuclear metal center, organophosphorus nerve agent, cobalt enzyme, Pyrococcus furiosus, biodecontamination The aims of this project were to structurally study and bioengineer thermostable prolidase from Pyrococcus furiosus to enable its use for oganophosphorus nerve agent detoxification. Prolidase contains one dinuclear Co metal-centermonomer and has optimal activity at 100 C, exhibiting no activity in the absence of Co2 or at temperatures 50 C. Requirement for metal ions is characteristic of all organophosphorus nerve agent hydrolases and results from these enzymes containing dinuclear metal-centers with one tight-binding metal atom and a second loose-binding metal atom. One primary objective of this study was to determine which of the metal sites is integral and which is labile, information that will be used to bioengineer prolidases. Another objective was to produce P. furiosus prolidase mutants that have increased catalytic activity over a lower range of temperatures using random mutation and a low-temperature selection method. Three mutant prolidases targeting metal-binding amino acids have been successfully produced and biochemical analysis has demonstrated that the Co1 metal-binding site is the high-affinity site and the Co2 site, the low-affinity site. Conditions for selection of mutant prolidases with increased activity at lower temperatures have been determined and mutant prolidases G39E and E236V isolated that have higher activity than wild type at 37 C.

Subject Categories:

  • Inorganic Chemistry
  • Organic Chemistry
  • Chemical, Biological and Radiological Warfare

Distribution Statement:

APPROVED FOR PUBLIC RELEASE