Improving the catalytic activity of hyperthermophilic Pyrococcus prolidases for detoxification of organophosphorus nerve agents over a broad range of temperatures.
Prolidase isolated from the hyperthermophilic archaeon Pyrococcus furiosus has potential for application for decontamination of organophosphorus compounds in certain pesticides and chemical warfare agents under harsh conditions. However, current applications that use an enzyme-based cocktail are limited by poor long-term enzyme stability and low reactivity over a broad range of temperatures. To obtain a better enzyme for OP nerve agent decontamination and to investigate structural factors that influence protein thermostability and thermoactivity, randomly mutated P. furiosus prolidases were prepared by using XL1-red-based mutagenesis and error-prone PCR. An Escherichia coli strain JD1 (lambdaDE3) (auxotrophic for proline [DeltaproA] and having deletions in pepQ and pepP dipeptidases with specificity for proline-containing dipeptides) was constructed for screening mutant P. furiosus prolidase expression plasmids. JD1 (lambdaDE3) cells were transformed with mutated prolidase expression plasmids and plated on minimal media supplemented with 50 muM Leu-Pro as the only source of proline. By using this positive selection, Pyrococcus prolidase mutants with improved activity over a broader range of temperatures were isolated. The activities of the mutants over a broad temperature range were measured for both Xaa-Pro dipeptides and OP nerve agents, and the thermoactivity and thermostability of the mutants were determined.
Theriot
Casey M
CM
Department of Microbiology, North Carolina State University, 4548 Gardner Hall, Campus Box 7615, Raleigh, NC 27695-7615, USA.
Du
Xuelian
X
Tove
Sherry R
SR
Grunden
Amy M
AM
D019943:Amino Acid Substitution / Q000235:genetics; D019843:Archaeal Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D002619:Chemical Warfare Agents / Q000378:metabolism*; D003470:Culture Media / Q000737:chemistry; D004150:Dipeptidases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D008958:Models, Molecular; D016296:Mutagenesis; D009943:Organophosphorus Compounds / Q000378:metabolism*; D010575:Pesticides / Q000378:metabolism*; D010957:Plasmids; D016133:Polymerase Chain Reaction / Q000379:methods; D055550:Protein Stability; D017434:Protein Structure, Tertiary; D020027:Pyrococcus furiosus / Q000201:enzymology* / Q000235:genetics; D017384:Sequence Deletion; D013696:Temperature
D019843:Archaeal Proteins; D002619:Chemical Warfare Agents; D003470:Culture Media; D009943:Organophosphorus Compounds; D010575:Pesticides; D004150:Dipeptidases; C022449:proline dipeptidase