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Dipeptidyl peptidase III from human symbiont Bacteroides thetaiotaomicron: isolation and characterization

The flow of data on primary structures of proteolytic enzymes enabled a new system of their classification based on evolutionary families. Peptidase family M49 (dipeptidyl peptidase III family) has been recently recognized among metallopeptidases, based on the unique structural motif, hexapeptide HEXXGH which harbors the predicted active site residues. The dipeptidyl peptidase III (DPP III), first discovered in the pituitary gland, was considered to be exclusively eukaryotic enzyme involved in intracellular peptide catabolism, with an implied role in defense against oxidative stress and pain-modulatory system. However, the new data of complete microbial genome sequences revealed in 2003 the first prokaryotic orthologs of M49 family, including putative DPP III from human gut symbiont Bacteroides thetaiotaomicron. Bacterial orthologs allowed us to define 5 evolutionary conserved sequences in proteins of DPP III family using bioinformatics. In order to investigate the properties of bacterial M49 peptidases, which show low homology with well-characterized mammalian enzymes, we overexpressed heterologously the full length cDNA encoding DPP III from bacterium B. thetaiotaomicron (675 amino acids) and purified it by a three-step procedure. Isolated bacterial DPP III was shown to be a monomeric acidic protein (Mr~72 000, pI 5.0-5.2) which preferred characteristic DPP III synthetic substrate Arg-Arg-2-naphthylamide. Comparison with the human counterpart revealed lower specific activity, pH optimum and stability of the bacterial DPP III, which seemed to be much more prone to oxidation. Both DPPs III were very sensitive to sulfhydryl reagent p-hydroxy-mercuribenzoate. This is the first report on the experimental characterization of the metallopeptidase M49 from bacteria. Further studies are needed to clarify the (nutritional) benefit which DPP III type of enzyme brings to our gut flora.

Dipeptidyl peptidase III ; Bacteroides thetaiotaomicron ; isolation ; characterization

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