OVEREXPRESSION AND CHARACTERIZATION OF A PROLYL ENDOPEPTIDASE FROM THE HYPERTHERMOPHILIC ARCHAEON PYROCOCCUS-FURIOSUS

The maltose-regulated mlr-2 gene from the hyperthermophilic archaeon P yrococcus furiosus having homology to bacterial and eukaryal prolyl en dopeptidase (PEPase) was cloned and overexpressed in Escherichia coli. Extracts from recombinant cells were capable of hydrolyzing the PEPas e substrate benzyloxycarbonyl-Gly-Pro-p-nitroanilide (ZGPpNA) with a t emperature optimum between 85 and 90 degrees C. Denaturing get electro phoresis of purified PEPase showed that enzyme activity was associated with a 70-kDa protein, which is consistent with that predicted from t he mlr-2 sequence. However, an apparent molecular mass of 59 kDa was o btained from gel permeation studies. In addition to ZGPpNA (K-Mapp of 53 mu M), PEPase was capable of hydrolyzing azocasein, although at a l ow rate. No activity was detected when ZGPpNA was replaced by substrat es for carboxypeptidase A and B, chymotrypsin, subtilisin, and neutral endopeptidase. (L-3-trans-Carboxirane-2-carbonyl)-L-Leu]-agmatine (E- 64) and tosyl-L-Lys chloromethyl ketone did not inhibit PEPase activit y. Both phenylmethylsulfonyl fluoride and diprotin A inhibited ZGPpNA cleavage, the latter doing so competitively (K-lapp of 343 mu M). At 1 00 degrees C, the enzyme displayed some tolerance to sodium dodecyl su lfate treatment, Stability of PEPase over time was dependent on protei n concentration; at temperatures above 65 degrees C, dilute samples re tained most of their activity after 24 h while the activity of concent rated preparations diminished significantly. This decrease was found t o be due, in part, to autoproteolysis. Partially purified PEPase from P. furiosus exhibited the same temperature optimum, molecular weight, and kinetic characteristics as the enzyme overexpressed in E. coli. Ex tracts from P. furiosus cultures grown in the presence of maltose were approximately sevenfold greater in PEPase activity than those grown w ithout maltose. Activity could not be detected in clarified medium obt ained from maltose-grown cultures. We conclude that mlr-2, now called prpA, encodes PEPase; the physiological role of this protease is prese ntly unknown.