Characterization of two proline dipeptidases (prolidases) from the hyperthermophilic archaeon Pyrococcus horikoshii.
Prolidases hydrolyze the unique bond between X-Pro dipeptides and can also cleave the P-F and P-O bonds found in organophosphorus compounds, including the nerve agents, soman and sarin. The advantages of using hyperthermophilic enzymes in biodetoxification strategies are based on their enzyme stability and efficiency. Therefore, it is advantageous to examine new thermostable prolidases for potential use in biotechnological applications. Two thermostable prolidase homologs, PH1149 and PH0974, were identified in the genome of Pyrococcus horikoshii based on their sequences having conserved metal binding and catalytic amino acid residues that are present in other known prolidases, such as the previously characterized Pyrococcus furiosus prolidase. These P. horikoshii prolidases were expressed recombinantly in the Escherichia coli strain BL21 (lambdaDE3), and both were shown to function as proline dipeptidases. Biochemical characterization of these prolidases shows they have higher catalytic activities over a broader pH range, higher affinity for metal and are more stable compared to P. furiosus prolidase. This study has important implications for the potential use of these enzymes in biotechnological applications and provides further information on the functional traits of hyperthermophilic proteins, specifically metalloenzymes.
Department of Microbiology, North Carolina State University, 4548 Gardner Hall, Campus Box 7615, Raleigh, NC 27695-7615, USA.
Casey M
CM
Theriot
Sherry R
SR
Tove
Amy M
AM
Grunden
D000595:Amino Acid Sequence; D019843:Archaeal Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D001709:Biotechnology / Q000379:methods*; D020134:Catalytic Domain; D003035:Cobalt / Q000378:metabolism; D004150:Dipeptidases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004795:Enzyme Stability; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D043902:Pyrococcus horikoshii / Q000201:enzymology* / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity
D019843:Archaeal Proteins; D011994:Recombinant Proteins; D003035:Cobalt; D004150:Dipeptidases; C022449:proline dipeptidase