Proline-specific endopeptidases from microbial sources: isolation of an enzyme from a Xanthomonas sp

Abstract

An extensive screening among microorganisms for the presence of post-proline-specific endopeptidase activity was performed. This activity was found among ordinary bacteria from soil samples but not among fungi and actinomycetes. This result is in contrast to the previous notion that this activity is confined to the genus Flavobacterium. A proline endopeptidase was isolated from a Xanthomonas sp. and characterized with respect to physicochemical and enzymatic properties. The enzyme is composed of a single peptide chain with a molecular weight of 75,000. The isoelectric point is 6.2. It is inhibited by diisopropylfluorophosphate and may therefore be classified as a serine endopeptidase. The activity profile is bell shaped with an optimum at pH 7.5.By using synthetic peptide substrates and intramolecular fluorescence quenching it was possible to study the influence of substrate structure on the rate of hydrolysis. The enzyme specifically hydrolyzed Pro-X peptide bonds. With Glu at position X, low rates of hydrolysis were observed; otherwise the enzyme exhibited little preference for particular amino acid residues at position X. A similar substrate preference was observed with respect to the amino acid residue preceding the prolyl residue in the substrate. The enzyme required a minimum of two amino acid residues toward the N terminus from the scissile bond, but further elongation of the peptide chain by up to six amino acid residues caused only a threefold increase in the rate of hydrolysis. Attempts to cleave at the prolyl residues in oxidized RNase failed, indicating that the enzyme does not hydrolyze long peptides, a peculiar property it shares with other proline-specific endopeptidases.Over the past 20 years a number of serine and thiol endopeptidases that cleave specifically on the carboxyl side of particular amino acid residues in peptides and proteins have been identified. These enzymes should be distinguished from the special-purpose endopeptidases (involved in, e.g., hormone processing and blood clotting) that recognize several amino acid residues in the substrate. Enzymes with specificity for cleavage at the carboxyl side of Asp and Glu (6,11,18), Glu (25, 31), Lys (13), Arg (7), Val (1), Gly (5), and Pro (2,10,20,21,23,34,(36)(37)(38)40) have been described. These enzymes are of interest from the point of view of understanding the nature of their high specificity, but they have also been used for specific cleavage of fusion proteins and for synthesis of peptide bonds.Due to the unusual structure of proline, most endopeptidases hydrolyze Pro-X bonds at extremely low rates; this has increased the interest in proline-specific enzymes. Such enzymes are commonly found in small quantities in plants (21, 37, 38) and in mammalian organs (2,10,20,23,29,34), where they appear to function in the degradation of numerous proline-containing peptide hormones (12,15,(26)(27)(28). In contrast, they have been reported to be rare in microorganisms; extensive screening only identified a single genus, Flavobacterium, with su...

Discovery, cloning and characterisation of proline specific prolyl endopeptidase, a gluten degrading thermo-stable enzyme from Sphaerobacter thermophiles

“…A number of studies have examined degradation of wheat gluten and to some extent barley gluten and components by prolyl endopeptidase initially extracted from Flavobacterium meningosepticum [15] and other microbes, such as Xanthomonas sp. [16], Aermonas hydrophilic [17], Sphingoonas capsulate [18], Halobacterium halobium S9 [19], Lactobacillus helveticus [20], Myxococcus…”

Portion-mixing peptide libraries of quenched fluorogenic substrates for complete subsite mapping of endoprotease specificity.

“…The donor-acceptor amino acid pair o-aminobenzamide (ABz)-3-nitrotyrosine [Tyr(NO2)] (13) for which excellent quenching of fluorescence is observed has recently been described (5). This donor-acceptor pair is conveniently introduced in parallel multiple-column peptide synthesis (MCPS) (14) of numerous substrates and has been used for subsite mapping of a variety of proteases (6,(15)(16)(17)(18)(19)(20)(21). However, substantial effort is still required to identify the optimal substrates.…”

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

“…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).…”