CLONING AND EXPRESSION OF A GENE ENCODING A BACTERIAL ENZYME FOR DECONTAMINATION OF ORGANOPHOSPHORUS NERVE AGENTS AND NUCLEOTIDE-SEQUENCE OF THE ENZYME

Organophosphorus acid (OPA) anhydrolase enzymes have been found in a w ide variety of prokaryotic and eukaryotic organisms. Interest in these enzymes has been prompted by their ability to catalyze the hydrolysis of toxic organophosphorus cholinesterase-inhibiting compounds, includ ing pesticides and chemical nerve agents. The natural substrates for t hese enzymes are unknown. The gene (opaA) which encodes an OPA. anhydr olase (OPAA-2) was isolated from an Alteromonas sp. strain JD6.5 EcoRI -lambda ZAPII chromosomal library expressed in Escherichia coil and id entified by immunodetection with anti-OPAA-2 serum. OPA anhydrolase ac tivity expressed by the immunopositive recombinant clones was demonstr ated by using diisopropylfluorophosphate (DFP) as a substrate. A compa rison of the recombinant enzyme with native, purified OPAA-2 showed th ey had the same apparent molecular mass (60 kDa), antigenic properties , and enzyme activity against DFP and the chemical nerve agents sarin, soman, and O-cyclohexyl methylphosphonofluoridate. The gene expressin g this activity aas found in a 1.74-kb PstI-HindIII fragment of the or iginal 6.1-kb EcoRI DNA insert. The nucleotide sequence of this PstI-H indIII fragment revealed an open reading frame of 1,551 nucleotides, c oding for a protein of 517 amino acid residues. Amino acid sequence co mparison of OPAA-2 with the protein database showed that OPAA-2 is sim ilar to a 647-amino-acid sequence produced by an open reading frame wh ich appears to be the E. coli pepQ gene. Further comparison of OPAA-2, the E. coli PepQ protein sequence, E. coli aminopeptidase P. and huma n prolidase showed regions of different degrees of similarity or funct ionally conserved amino acid substitutions. These findings, along with preliminary data confirming the presence of prolidase activity expres sed by OPAA-2, suggest that the OPAA-2 enzyme may, in nature, be used in peptide metabolism.