Overexpression and characterization of a prolyl endopeptidase from the hyperthermophilic archaeon Pyrococcus furiosus

Abstract

The maltose-regulated mlr-2 gene from the hyperthermophilic archaeon Pyrococcus furiosus having homology to bacterial and eukaryal prolyl endopeptidase (PEPase) was cloned and overexpressed in Escherichia coli. Extracts from recombinant cells were capable of hydrolyzing the PEPase substrate benzyloxycarbonyl-GlyPro-p-nitroanilide (ZGPpNA) with a temperature optimum between 85 and 90°C. Denaturing gel electrophoresis of purified PEPase showed that enzyme activity was associated with a 70-kDa protein, which is consistent with that predicted from the mlr-2 sequence. However, an apparent molecular mass of 59 kDa was obtained from gel permeation studies. In addition to ZGPpNA (K Mapp of 53 M), PEPase was capable of hydrolyzing azocasein, although at a low rate. No activity was detected when ZGPpNA was replaced by substrates for carboxypeptidase A and B, chymotrypsin, subtilisin, and neutral endopeptidase. N-[N-(L-3-trans-Carboxirane-2-carbonyl)-L-Leu]-agmatine (E-64) and tosyl-L-Lys chloromethyl ketone did not inhibit PEPase activity. Both phenylmethylsulfonyl fluoride and diprotin A inhibited ZGPpNA cleavage, the latter doing so competitively (K lapp of 343 M). At 100°C, the enzyme displayed some tolerance to sodium dodecyl sulfate treatment. Stability of PEPase over time was dependent on protein concentration; at temperatures above 65°C, dilute samples retained most of their activity after 24 h while the activity of concentrated preparations diminished significantly. This decrease was found to 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. Extracts from P. furiosus cultures grown in the presence of maltose were approximately sevenfold greater in PEPase activity than those grown without maltose. Activity could not be detected in clarified medium obtained from maltose-grown cultures. We conclude that mlr-2, now called prpA, encodes PEPase; the physiological role of this protease is presently unknown.Pyrococcus furiosus is an obligately anaerobic marine archaeon whose optimal growth temperature is 100°C (11). The organism grows on a variety of complex carbon sources including starch and peptides and is capable of reducing sulfur (3,11,15,29). Because of its hyperthermophilic characteristics, P. furiosus has been the focus of significant biochemical and physiological studies and is one of the most studied hyperthermophilic archaea to date (1, 15, 30). Several proteins have been shown to function at temperatures greater than 90°C (15, 17), and enzymes displaying novel activities have been isolated and characterized (1,12,18). A few classes of hydrolytic enzymes from P. furiosus and other hyperthermophiles, including those having proteolytic activities, have been examined (1,6,10,12,17). P. furiosus produces several proteases with estimated molecular masses ranging from 66 to 135 kDa (2, 6, 10, 32). These proteases are highly stable at temperatures of 95°C and abov...

Structure and Catalysis of Acylaminoacyl Peptidase

“…However, several papers have indicated that some serine oligopeptidases hydrolyze large proteins. For example, fibroblast activation protein has gelatinase and collagenase activities (51), oligopeptidase B cleaves at a restricted site of histone proteins (52), and POP digests itself (53,54) or degrades a splice variant protein of p40-phox (55). In all of these cases the hydrolytic reactions require unusually long times, at least overnight.…”

Kinetic and Mechanistic Studies of Prolyl Oligopeptidase from the Hyperthermophile Pyrococcus furiosus

“…Mechanistic and structural studies of the ␤-glucosidase (2) and glutamate dehydrogenase (3,4) from P. furiosus have demonstrated the similarities between enzyme homologues from organisms with very different temperature optima, and structural data obtained for mesophilic proteins can be useful for predicting the structure of hyperthermostable proteins. Recently, the gene for the prolyl oligopeptidase of P. furiosus was cloned and overexpressed in Escherichia coli as a functional protease (5). The recombinant prolyl oligopeptidase displayed characteristics identical to those of the enzyme expressed in P. furiosus (5), affording the opportunity to readily obtain sufficient quantities of this protein for kinetic studies.…”

“…Recently, the gene for the prolyl oligopeptidase of P. furiosus was cloned and overexpressed in Escherichia coli as a functional protease (5). The recombinant prolyl oligopeptidase displayed characteristics identical to those of the enzyme expressed in P. furiosus (5), affording the opportunity to readily obtain sufficient quantities of this protein for kinetic studies.…”

“…In fact, the order of the amino acids in the primary structure is different for prolyl oligopeptidase (Ser, Asp, His) than it is for either the chymotrypsin (His, Asp, Ser) or subtilisin (Asp, His, Ser) families (9). Prolyl oligopeptidases (POPs) 1 were first isolated from human tissue (10) and subsequently from the tissues of other mammals (11,12), fungi (13,14), bacteria, (15)(16)(17), and archaea (5).…”

Pseudomurein endoisopeptidases PeiW and PeiP, two moderately related members of a novel family of proteases produced in Methanothermobacter strains

“…from Pyrococcus kodakaraensis KOD1 [5] ; (iv) a maltose-regulated prolyl oligopeptidase from Pyrococcus furiosus [6]; (v) metalloproteases such as S. solfataricus zinc carboxypeptidase [7] and P. furiosus cobalt-activated carboxypeptidase [8]. In addition, a number of yet unclassi¢ed archaeal proteases were also reported, such as Thermus aquaticus caldolysin [9].…”