Characterization of two proline dipeptidases (prolidases) from the hyperthermophilic archaeon Pyrococcus horikoshii

Abstract

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.

The Order Thermococcales and the Family Thermococcaceae

“…litoralis The Order Thermococcales and the Family Thermococcaceae . Tsunasawa et al (1998) Prolidase (proline dipeptidase) P. furiosus Ghosh et al (1998) P. horikoshii Theriot et al (2009Theriot et al ( , 2010 locations (see > Table 27.2). They are found all around the globe where hydrothermal are present (Lepage et al 2004).…”

“…(2004) The Order Thermococcales and the Family Thermococcaceae Theriot et al 2009Theriot et al , 2010Ward et al 2002b). The derived amino acids are oxidized to their corresponding organic acids with the generation of ATP by substrate-level phosphorylation.…”

Improving the Catalytic Activity of HyperthermophilicPyrococcus horikoshiiProlidase for Detoxification of Organophosphorus Nerve Agents over a Broad Range of Temperatures

“…In response to the need to develop stable OP nerve agent degrading enzyme systems, thermostable prolidases from Pyrococcus spp. were studied [16–18]. …”

“…Specifically, Pyrococcus prolidases from P. furiosus (PF1343 or Pf prol) and P. horikoshii (PH1149 or Ph prol and PH0974 or Ph1 prol) were characterized both structurally and enzymatically [16, 18, 19]. The Pyrococcus prolidases were determined to be very similar, with Pf prol showing 88% amino acid similarity to Ph prol and 55% similarity to Ph1 prol.…”

“…Ph1 prol has demonstrated higher activity at lower temperatures and over a broader pH range. It has long-term stability, higher affinity for substrates, and a lower metal requirement for catalysis [16]. Therefore, it was deemed to be advantageous to use Ph1 prol in mutagenesis studies in order to develop a better enzyme for OP nerve agent detoxification and to further investigate factors that influence the catalysis of thermophilic metalloenzymes.…”