Aminopeptidase from a thermophilic strain of Bacillus licheniformis

Aminopeptidase is isolated and purified from the culture liquid of the thermophilic strain of Bacillus licheniformis. The aminopeptidase predominantly splits off N-terminal leucin in short peptides and hydrolyzes leucinamide as well. The molecular weight of the enzyme is about 60 kDa. The enzyme is able to form aggregates. Optimum of aminopeptidase activity was demonstrated at pH 8.0-8.3 and temperature of 85 degrees C. The enzyme is inactivated by metal-binding reagents and reducing substances, and is activated by cobalt and PCMB ions. The EDTA-inactivated enzyme activity is reduced by cobalt and zinc ions, however the latter has no activating action. The enzyme under study is characterized by high thermostability: in the presence of the substrate at the temperature of 90 degrees C the reaction linearity is retained for not less than 2 h and without the substrate the half-life of the aminopeptidase at 90 degrees C is 145 min. Extracellular aminopeptidase of the thermophilic strain of B. licheniformis is a new enzyme differing from the aminopeptidases described by the present in high thermostability, induced, evidently, by the presence of one or several disulphide bonds in the enzyme molecule.     

Novel prolyl tri/tetra-peptidyl aminopeptidase from Streptomyces mobaraensis: substrate specificity and enzyme gene cloning

The prolyl peptidase that removes the tetra-peptide of pro-transglutaminase was purified from Streptomyces mobaraensis mycelia. The substrate specificity of the enzyme using synthetic peptide substrates showed proline-specific activity with not only tripeptidyl peptidase activity, but also tetrapeptidyl peptidase activity. However, the enzyme had no other exo- and endo-activities. This substrate specificity is different from proline specific peptidases so far reported. The enzyme gene was cloned, based on the direct N-terminal amino acid sequence of the purified enzyme, and the entire nucleotide sequence of the coding region was determined. The deduced amino acid sequence revealed an N-terminal signal peptide sequence (33 amino acids) followed by the mature protein comprising 444 amino acid residues. This enzyme shows no remarkable homology with enzymes belonging to the prolyl oligopeptidase family, but has about 65% identity with three tripeptidyl peptidases from Streptomyces lividans, Streptomyces coelicolor, and Streptomyces avermitilis. Based on its substrate specificity, a new name, "prolyl tri/tetra-peptidyl aminopeptidase," is proposed for the enzyme.     

Structural and immunological evidence for the identity of prolyl aminopeptidase with leucyl aminopeptidase

Prolyl aminopeptidase (EC 3.4.11.5) has been assumed to be a unique enzyme catalyzing specifically the removal of unsubstituted NH2-terminal L-prolyl residues from various peptides and to be distinct from leucyl aminopeptidase (EC 3.4.11.1). In the present study, prolyl aminopeptidases were purified to apparent homogeneity from pig small intestine mucosa and human liver and their NH2-terminal amino acid sequences were determined together with that of pig kidney leucyl aminopeptidase. The NH2-terminal 24-residue sequence of pig intestinal prolyl aminopeptidase was shown to be identical with that of pig kidney leucyl aminopeptidase. The NH2-terminal sequence of human liver prolyl aminopeptidase was also shown to be very similar to that of pig kidney leucyl aminopeptidase. Further, pig intestinal prolyl aminopeptidase and pig kidney leucyl aminopeptidase were immunologically indistinguishable. These lines of evidence strongly suggest that prolyl aminopeptidase is identical with leucyl aminopeptidase.     

Proline residues in the maturation and degradation of peptide hormones and neuropeptides

The proteases involved in the maturation of regulatory peptides like those of broader specificity normally fail to cleave peptide bonds linked to the cyclic amino acid proline. This generates several mature peptides with N-terminal X-Pro-sequences. However, in certain non-mammalian tissues repetitive pre-sequences of this type are removed by specialized dipeptidyl (amino)peptidases during maturation. In mammals, proline-specific proteases are not involved in the biosynthesis of regulatory peptides, but due to their unique specificity they could play an important role in the degradation of them. Evidence exists that dipeptidyl (amino)peptidase IV at the cell surface of endothelial cells sequesters circulating peptide hormones which are then susceptible to broader aminopeptidase attack. The cleavage of several neuropeptides by prolyl endopeptidase has been demonstrated in vitro, but its role in the brain is questionable since the precise localization of the protease is not clarified.     

PepS from Streptococcus thermophilus. A new member of the aminopeptidase T family of thermophilic bacteria.

The proteolytic system of lactic acid bacteria is essential for bacterial growth in milk but also for the development of the organoleptic properties of dairy products. Streptococcus thermophilus is widely used in the dairy industry. In comparison with the model lactic acid bacteria Lactococcus lactis, S. thermophilus possesses two additional peptidases (an oligopeptidase and the aminopeptidase PepS). To understand how S. thermophilus grows in milk, we purified and characterized this aminopeptidase. PepS is a monomeric metallopeptidase of approximately 45 kDa with optimal activity in the range pH 7.5-8.5 and at 55 degrees C on Arg-paranitroanilide as substrate. PepS exhibits a high specificity towards peptides possessing arginine or aromatic amino acids at the N-terminus. From the N-terminal protein sequence of PepS, we deduced degenerate oligonucleotides and amplified the corresponding gene by successive PCR reactions. The deduced amino-acid sequence of the PepS gene has high identity (40-50%) with the aminopeptidase T family from thermophilic and extremophilic bacteria; we thus propose the classification of PepS from S. thermophilus as a new member of this family. In view of its substrate specificity, PepS could be involved both in bacterial growth by supplying amino acids, and in the development of dairy products' flavour, by hydrolysing bitter peptides and liberating aromatic amino acids which are important precursors of aroma compounds.     

Characterisation of <Emphasis Type="Italic">Aspergillus niger</Emphasis> prolyl aminopeptidase

We have cloned a gene (papA) that encodes a prolyl aminopeptidase from Aspergillus niger. Homologous genes are present in the genomes of the Eurotiales A. nidulans, A. fumigatus and Talaromyces emersonii, but the gene is not present in the genome of the yeast Saccharomyces cerevisiae. Cell extracts of strains overexpressing the gene under the control of its own promoter showed a fourfold to sixfold increase in prolyl aminopeptidase activity, but no change in phenylalanine or leucine aminopeptidase activity. The overexpressed enzyme was subsequently purified and characterised. The enzyme specifically removes N-terminal proline and hydroxyproline residues from peptides. It is the first enzyme of its kind from a eukaryotic organism that has been characterised.     

Purification and properties of a novel glycine amino peptidase from Actinomucor elegans and its potential application

AIMS: To study the properties and show the potential application of a glycine aminopeptidase from Actinomucor elegans. METHODS AND RESULTS: The enzyme was estimated to have molecular mass of 320 kDa by gel filtration and the subunit size of 56.5 kDa by SDS-PAGE. It hydrolysed glycine from substrate more efficiently than other amino acids. The optimal temperature for this enzyme was 40 degrees C and at pH 8.0 it showed its highest activity. The Km and Kcat of the enzyme for glycine-beta-naphthylamine was 0.24 mm and 100.8 s(-1), respectively. Zinc, copper, cadmium and o-phenanthrolin suppressed almost all enzyme activities at the concentration of 1.0 mm. In the process of hydrolysing proteins, it could improve the protease activity considerably. CONCLUSIONS: It was a hexamer metalloenzyme which was specific for the substrates with glycinse residue at the N-terminal and some metal cations were needed to maintain its activity. SIGNIFICANCE AND IMPACT OF THE STUDY: This study demonstrates the properties of a novel aminopeptidase and shows its potential application in the process of the food industry.     

Localization of collagen prolyl hydroxylase to the hepatocyte : Studies in primary monolayer cultures of parenchymal cells from adult rat liver

Significant levels of prolyl hydroxylase activity (prolyl-glycyl-peptide, 2-oxoglutarate: oxygen oxidoreductase; EC 1.14.11.2) have been found in freshly isolated hepatocytes prepared from normal or regenerated adult rat liver and primary non-proliferating monolayer cultures of these cells. Four days after partial hepatectomy, the intact regenerated liver contained two times the normal level of prolyl hydroxylase activity. Freshly isolated hepatocytes contained 24% of the total prolyl hydroxylase activity in normal liver and 47% of that in regenerated liver. Upon incubation of hepatocytes for 24 h in a chemically defined culture medium containing insulin, prolyl hydroxylase activity rose 2- to 3-fold, and gradually declined during the next 48 h. The rise in prolyl hydroxylase activity was blocked by addition of cycloheximide to the culture medium. The presence of prolyl hydroxylase activity in hepatocyte cultures was not likely due to contamination with non-parenchymal liver cells. The latter cells contained less than 20% of the total enzyme activity recovered in all cells isolated from the liver. Furthermore, prolyl hydroxylase was localized by immunofluorescence uniformly to the hepatocytes in culture. Cultured hepatocytes converted [¹⁴C]proline to [¹⁴C]hydroxyproline at rates comparable to those reported for whole liver. However, only a small portion of the hydroxyproline containing product was present as collagen protein, suggesting its rapid degradation in culture. We conclude that the liver parenchymal cell may actively participate in collagen synthesis and possibly in collagen degradation.     

The purification and characterization of a proline dipeptidase from guinea pig brain

A proline dipeptidase (EC 3.4.13.9) from guinea pig brain was purified to over 90% homogeneity by a combination of ammonium sulfate fractionation, DEAE-cellulose chromatography, calcium phosphate-cellulose chromatography, chromatofocusing, and gel filtration on Sephadex G-200. A purification factor of 2718-fold was obtained with a yield of 7%. The purified enzyme was found to have an apparent molecular weight of 132,000 and to consist of two dissimilar subunits of molecular weights 64,000 and 68,000. The substrate specificity of the enzyme is not that of a strict proline dipeptidase. Although it preferentially hydrolyzes proline dipeptides (Leu-Pro) it also hydrolyzes prolyl dipeptides (Pro-Leu) and dipeptides not containing proline (Leu-Leu). The purified enzyme preparation exhibited weak aminoacylproline aminopeptidase activity against Arg-Pro-Pro but it did not exhibit any post-proline dipeptidyl aminopeptidase, post-proline cleaving endopeptidase, proline iminopeptidase, prolyl carboxypeptidase or carboxypeptidase P activities when tested with a large variety of peptides and arylamides. With all of the proline and prolyl dipeptides examined the enzyme exhibited biphasic kinetics (two distinct slopes on Lineweaver-Burk plots). However, with Leu-Leu as substrate normal Michaelis-Menten kinetics were obeyed.     

Degradation of proline peptides in peptidase-deficient strains of Salmonella typhimurium.

A mutant strain of Salmonella typhimurium that lacks two proline-specific peptidases (peptidases P and Q) could not complete the degradation of proline peptides formed as intermediates in starvation-induced protein breakdown. The wild-type strain produced free proline as the product of degradation of proline-labeled proteins. The pepP pepQ mutant, however, produced a mixture of small proline peptides. In the absence of peptidase Q only, peptidase P could complete the degradation of most of the proline peptide intermediates formed. In the absence of peptidase P only, about 50% of the proline-labeled, acid-soluble products were proline peptides. These results are consistent with in vitro specificity data indicating that peptidase Q hydrolyzes X-Pro dipeptides only, whereas peptidase P attacks both X-Pro dipeptides and longer peptides with X-Pro at their N-termini. A mutant strain lacking four broad-specificity peptidases (peptidases N, A, B, and D), but containing peptidases P and Q, also produced proline peptides as products of protein breakdown. This observation suggests that broad-specificity peptidases are required to generate the X-Pro substrates of peptidases P and Q. A strain lacking six peptidases (N, A, B, D, P, and Q) was constructed and produced less free proline from protein breakdown than either the pepP pepQ strain or the pepN pepA pepB pepD strain. These observations suggest that the degradation of peptide intermediates involves the sequential removal of N-terminal amino acids and requires both broad-specificity aminopeptidases (peptidases N, A, and B) and the X-Pro-specific aminopeptidase, peptidase P.     

Structure of human cytosolic X-prolyl aminopeptidase: a double Mn(II)-dependent dimeric enzyme with a novel three-domain subunit

X-prolyl aminopeptidases catalyze the removal of a penultimate prolyl residue from the N termini of peptides. Mammalian X-prolyl aminopeptidases are shown to be responsible for the degradation of bradykinin, a blood pressure regulator peptide, and have been linked to myocardial infarction. The x-ray crystal structure of human cytosolic X-prolyl aminopeptidase (XPN-PEP1) was solved at a resolution of 1.6 angstroms. The structure reveals a dimer with a unique three-domain organization in each subunit, rather than the two domains common to all other known structures of X-prolyl aminopeptidase and prolidases. The C-terminal catalytic domain of XPNPEP1 coordinates two metal ions and shares a similar fold with other prolyl aminopeptidases. Metal content analysis and activity assays confirm that the enzyme is double Mn(II) dependent for its activity, which contrasts with the previous notion that each XPNPEP1 subunit contains only one Mn(II) ion. Activity assays on an E41A mutant demonstrate that the acidic residue, which was considered as a stabilizing factor in the protonation of catalytic residue His498, plays only a marginal role in catalysis. Further mutagenesis reveals the significance of the N-terminal domain and dimerization for the activity of XPNPEP1, and we provide putative structural explanations for their functional roles. Structural comparisons further suggest mechanisms for substrate selectivity in different X-prolyl peptidases.     

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

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.     

Immunochemical characterization of a proline endopeptidase from rat brain. Its relationship to proline endopeptidase from other tissues and from other species

Monospecific antiserum raised against rat brain proline endopeptidase is used to demonstrate the ubiquity of the enzyme and its unique role in the degradation of proline-containing peptides. All endoproteolytic activity directed toward proline residues in several rat tissues is shown to share one or more common antigenic determinants with rat brain proline endopeptidase. Similar activity from tissue of other species crossreacts with rat proline endopeptidase. The data presented suggest that proline endopeptidase is the sole cytoplasmic enzyme capable of degrading proline-containing peptides in every tissue examined and that previously reported proline-specific endoproteolytic activities observed in a variety of systems may be ascribed to proline endopeptidase. The putative role of proline endopeptidase in protein degradation is discussed.     

Physiology and enzymology of thermophilic anaerobic bacteria degrading starch

Abstract The capability of secreting thermoactive enzymes exhibiting α-amylase and pullulanase with debraching activity, seems to be widely distributed amongst anaerobic thermophilic bacteria. Interestingly, pullulanase formed by these bacteria displays dual specificity by attacking α-1,6- as well as α-1,4-glycosidic linkages in branched glucose polymers. Unlike the enzyme system of aerobic microorganisms the majority of starch hydrolysing enzymes of anaerobic bacteria is metal indepedent and is extremely thermostable. This enzyme system is controlled by substrate induction and catabolite repression; enzyme expression is accomplished when maltose or maltose-containing carbohydrates are used as substrates. By developing a process in continuous culture we were able to greatly enhance enzyme synthesis and release by anaerobic thermophilic bacteria. An elevation in the specific activities of cell-free amylases and pullulanases could also be achieved by entrapping of bacteria in calcium alginate beads. The unique properties of extracellular enzymes of thermophilic anaerobic bacteria makes this group of organisms suitable candidates for inductrial application.     

In vitro metabolism of LVV-Hemorphin-7 by renal cytosol and purified prolyl endopeptidase

The metabolism of LVVH7, an endogenous peptide obtained by cathepsin D hydrolysis of the beta chain of hemoglobin, was studied, in vitro, in the presence of cytosol of rat kidney and compared with angiotensin IV. High metabolic activity was found against these two peptides (half life time < 2 min) in this subcellular fraction. The main products of LVVH7 metabolism by renal cytosol are VVH7, H7 and LVVH6 suggesting both aminopeptidase and carboxypeptidase activities. The use of PEP inhibitor in kidney cytosol permitted to demonstrate the major role of prolyl endopeptidase (PEP) in LVVH7 degradation. This fact was reinforced by a kinetic study investigated with purified enzyme (Km/Vmax about 238 mM-1 s-1 and close to that observed for angiotensin related peptides).     

Estimates of leucine aminopeptidase activity in different marine and brackish environments

AIM: Leucine aminopeptidase (LAP), an enzyme involved in the decomposition of natural peptides, was measured in different marine and brackish ecosystems, together with some environmental and microbiological parameters. METHODS AND RESULTS: The fluorogenic compound L-leucine-7-amido-4-methyl coumarin was specifically used for the determination of this in situ activity. The enzyme data obtained from this comparative study highlighted the strong spatial and temporal variability of the distribution of LAP in aquatic ecosystems, which was sometimes related to the course of environmental variables such as salinity and organic carbon content. CONCLUSIONS: LAP assay has proved to be a rapid method providing useful information on the microbial metabolic processes involved in the mineralization of organic matter. SIGNIFICANCE AND IMPACT OF THE STUDY: The determination of the potential rates of extracellular enzyme activity is of great ecological importance to extend knowledge on the role played by bacteria in aquatic biogeochemical cycles.     

Prolyl aminopeptidase from rat brain and kidney. Action on peptides and identification as leucyl aminopeptidase

Based on the liberation of proline from ProLeuGlyNH2 (MIF-1, melanostatin) manganese-activated prolyl aminopeptidase activities were purified from rat brain and kidney cytosolic fractions. They were distinguished from other di- and tripeptidases and an arylamidase liberating N-terminal proline. Purified prolyl aminopeptidase from both sources had identical molecular properties (native Mr 300,000, subunit Mr 54,000) and very similar catalytic properties. The action of the purified enzymes was not restricted to proline. Other, in particular lipophilic, amino acids were cleaved from di-, tri- and oligopeptides with even higher velocities. Peptides with N-terminal penultimate proline residues were not degraded. From a comparison of molecular data, action on peptides, influence of pH values, inhibitors and activators, it is concluded that the activity is identical with leucyl aminopeptidase (EC 3.4.11.1) and that a separate prolyl aminopeptidase (EC 3.4.11.5) does not exist in rats.     

Studies on the mechanism of reduction of prolyl hydroxylase activity by D,L-3,4 dehydroproline.

When chick frontal bone cells in culture were exposed to d,l-3,4 dehydroproline, the specific activity of prolyl hydroxylase was markedly reduced, but the concentration of the protein antigenically related to prolyl hydroxylase was not decreased. The specific activity of purified prolyl hydroxylase from cells grown in d,l-3,4 dehydroproline was significantly lower than that of control cells. Preincubation of a homogeneous preparation of chick embryo prolyl hydroxylase with collagenous peptides containing [¹⁴C]d,l-3,4 dehydroproline resulted in a time-dependent decrease in the enzymatic activity. These observations suggest that the in vivo reduction in prolyl hydroxylase activity by dehydroproline could be either due to an interaction of the enzyme with collagenous peptides containing dehydroproline and/or the synthesis of an aberrant form of prolyl hydroxylase with decreased enzymatic activity.     

Peptidases of the rumen bacterium, Prevotella ruminicola

Prevotella (formerly Bacteroides) ruminicola is a numerous rumen bacterium which plays a significant role in the metabolism of proteins and peptides in the rumen. Measurement of the hydrolysis of synthetic aminopeptidase substrates by sonicated extracts and whole cells of different species of rumen bacteria indicated that P. ruminicola had the greatest range and specific activity of dipeptidyl peptidases among the species tested.Streptococcus bovis hydrolysed some dipeptidyl peptidase substrates to a lesser extent, and several species broke down Ala2-p-nitroanilide, including Ruminobacter amylophilus, Ruminococcus spp. and Veillonella parvula. Dipeptidyl peptidases, which cleave dipeptides from the amino-terminus of longer peptides, were much more active than aminopeptidases removing single amino acids in P. ruminicola. Ion-exchange chromatography of sonicated extracts of P. ruminicola M384 revealed at least four distinct activities: one hydrolysed Ala2-p-nitroanilide, ValAla-p-nitroanilide, Ala4and Ala5; another was an O2-sensitive activity hydrolysing GlyArg-4-methoxynapthylamide, ArgArg-4-methoxynaphthylamide, Gly5 and ValGlySerGlu, similar to dipeptidyl peptidase type I DPP-1); a third hydrolysed GlyPro-p-nitroanilide and GlyPro-4-methoxynapthylamide and was similar to dipeptidyl peptidase type IV XDPP-4); a fourth broke down LysAla-4-methoxynaphthylamide. All of the enzymes, and particularly those active against Ala2-p-nitroanilide and GlyPro-p-nitroanilide, were inhibited by serine protease inhibitors, and all except DPP-4 were inhibited by EDTA. Both DPP-1 and the enzyme hydrolysing LysAla-4-methoxynaphthylamide were inhibited strongly by iodoacetate. DPP-4 was inhibited completely by diprotin A. Competitive inhibition experiments suggested that DPP-1 was less important than the other enzymes in the breakdown of peptide mixtures.     

Thermostable aminopeptidase from Pyrococcus horikoshii

From the genome sequence data of the thermophilic archaeon Pyrococcus horikoshii, an open reading frame was found which encodes a protein (332 amino acids) homologous with an endoglucanase from Clostridium thermocellum (42% identity), deblocking aminopeptidase from Pyrococcus furiosus (42% identity) and an aminopeptidase from Aeromonas proteolytica (18% identity). This gene was cloned and expressed in Escherichia coli, and the characteristics of the expressed protein were examined. Although endoglucanase activity was not detected, this protein was found to have aminopeptidase activity to cleave the N-terminal amino acid from a variety of substrates including both N-blocked and non-blocked peptides. The enzyme was stable at 90 degrees C, with the optimum temperature over 90 degrees C. The metal ion bound to this enzyme was calcium, but it was not essential for the aminopeptidase activity. Instead, this enzyme required the cobalt ion for activity. This enzyme is expected to be useful for the removal of N(alpha)-acylated residues in short peptide sequence analysis at high temperatures.