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PubMed1986

Active-site-serine D-alanyl-D-alanine-cleaving-peptidase-catalysed acyl-transfer reactions. Procedures for studying the penicillin-binding proteins of bacterial plasma membranes.

Under certain conditions, the values of the parameters that govern the interactions between the active-site-serine D-alanyl-D-alanine-cleaving peptidases and both carbonyl-donor substrates and beta-lactam suicide substrates can be determined on the basis of the amounts of (serine ester-linked) acyl-protein formed during the reactions. Expressing the 'affinity' of a beta-lactam compound for a DD-peptidase in terms of second-order rate constant of enzyme acylation and first-order rate constant of acyl-enzyme breakdown rests upon specific features of the interaction (at a given temperature) and permits study of structure-activity relationships, analysis of the mechanism of intrinsic resistance and use of a 'specificity index' to define the capacity of a beta-lactam compound of discriminating between various sensitive enzymes. From knowledge of the first-order rate constant of acyl-enzyme breakdown and the given time of incubation, the beta-lactam compound concentrations that are necessary to achieve given extents of DD-peptidase inactivation can be converted into the second-order rate constant of enzyme acylation. The principles thus developed can be applied to the study of the multiple penicillin-binding proteins that occur in the plasma membranes of bacteria.

Acylation↗
PubMed1990

Thiol-dependent metallo-endopeptidase characteristics of Pz-peptidase in rat and rabbit.

Pz-peptidase was purified from rat testis and rabbit muscle. Zinc was detectable in the rat enzyme. The activity of the enzyme from both species was slowly but completely abolished by EDTA and restored by Zn2+. Free thiol groups were also important for the catalytic activity of rat Pz-peptidase, as previously reported for the rabbit enzyme. We conclude that in both species Pz-peptidase has the characteristics of a thiol-dependent metallo-endopeptidase.

Amino Acid Sequence↗
PubMed1991

A quenched fluorescent substrate for thimet peptidase containing a new fluorescent amino acid, DL-2-amino-3-(7-methoxy-4-coumaryl)propionic acid.

DL-2-Amino-3-(7-methoxy-4-coumaryl)propionic acid, a new fluorescent amino acid (abbreviated to Amp), has been synthesized to provide an alternative to tryptophan in quenched fluorescent peptide substrates for peptidases. The model compound Ac-DL-Amp-NH2 was intensely fluorescent with an excitation maximum at 328 nm and an emission maximum at 392 nm. Fmoc (fluoren-9-ylmethoxycarbonyl)-DL-Amp was made to allow the solid-phase synthesis of Amp-containing peptides by the Fmoc-polyamide method. The peptide derivative Dnp (2,4-dinitrophenyl)-Pro-Leu-Gly-Pro-DL-Amp-D-Lys was cleaved by thimet peptidase at the Leu-Gly bond, with a 20-fold enhancement of fluorescence. The value of kcat./Km for thimet peptidase was 6.7 x 10(5) M-1.s-1, compared with the value of 2.4 x 10(5) M-1.s-1 for the tryptophan-containing analogue, Dnp-Pro-Leu-Gly-Pro-Trp-D-Lys.

Alanine↗
PubMed1992

Primary and predicted secondary structures of the Actinomadura R39 extracellular DD-peptidase, a penicillin-binding protein (PBP) related to the Escherichia coli PBP4.

As derived from gene cloning and sequencing, the 489-amino-acid DD-peptidase/penicillin-binding protein (PBP) produced by Actinomadura R39 has a primary structure very similar to that of the Escherichia coli PBP4 [Mottl, Terpstra & Keck (1991) FEMS Microbiol. Lett. 78, 213-220]. Hydrophobic-cluster analysis of the two proteins shows that, providing that a large 174-amino-acid stretch is excluded from the analysis, the bulk of the two polypeptide chains possesses homologues of the active-site motifs and secondary structures found in the class A beta-lactamase of Streptomyces albus G of known three-dimensional structure. The 174-amino-acid insert occurs at equivalent places in the two PBPs, between helices alpha 2 and alpha 3, away from the active site. Such an insert is unique among the penicilloyl serine transferases. It is proposed that the Actinomadura R39 PBP and E. coli PBP4 form a special class, class C, of low-Mr PBPs/DD-peptidases. A vector has been constructed and introduced by electrotransformation in the original Actinomadura R39 strain, allowing high-level expression and secretion of the DD-peptidase/PBP (250 mg.l-1). The gene encoding the desired protein is processed differently in Actinomadura R39 and Streptomyces lividans. Incorrect processing in Streptomyces lividans leads to a secreted protein which is inert in terms of DD-peptidase activity and penicillin-binding capacity.

Actinomycetales↗
PubMed1993

Evolutionary families of peptidases.

The available amino acid sequences of peptidases have been examined, and the enzymes have been allocated to evolutionary families. Some of the families can be grouped together in 'clans' that show signs of distant relationship, but nevertheless, it appears that there may be as many as 60 evolutionary lines of peptidases with separate origins. Some of these contain members with quite diverse peptidase activities, and yet there are some striking examples of convergence. We suggest that the classification by families could be used as an extension of the current classification by catalytic type.

Amino Acid Sequence↗
PubMed1993

Processing of chimeric mammalian cytochrome b5 precursors in Escherichia coli: reaction specificity of signal peptidase and identification of an aminopeptidase in post-translocational processing.

A chimeric precursor interlinked by an arginine residue between the full-length signal sequence of alkaline phosphatase and the eukaryotic cytoplasmic cytochrome b5 was constructed. Expression of the chimeric precursor protein in Escherichia coli resulted in efficient export of spectrally authentic cytochrome b5 into the periplasm [Karim, Harding, Evans, Kaderbhai and Kaderbhai (1993) Bio/Technology 11, 612-618]. On sequencing, the apparent absence of arginine at the N-terminus of the secreted cytochrome b5 implied that the chimera was either miscleaved by signal peptidase or further processed following signal excision by an uncharacterized peptidase. The influence of the N-terminal region of cytochrome b5 on the unusual processing of the chimeric precursor was investigated by engineering a number of variant forms in which the region between Arg+1 and the mature portion of cytochrome b5 was extended and varied. Observations of the in vivo processed patterns of these variant cytochrome b5 forms exported into the periplasm revealed that the absence of arginine was due to neither miscleavage of the translocated precursor by the signal peptidase nor the nature of the early region of cytochrome b5. In fact, the selective excision of the arginine residue occurred subsequent to signal sequence deletion by an aminopeptidase which was sensitive to the metal chelator o-phenanthroline. We show that this aminopeptidase also participates in the trimming of the N-terminal arginine residue of the bacterial alkaline phosphatase to generate the three isoenzymes in the periplasm.

Amino Acid Sequence↗
PubMed2002

Identification and properties of type I-signal peptidases of Bacillus amyloliquefaciens.

The use of Bacillus amyloliquefaciens for enzyme production and its exceptional high protein export capacity initiated this study where the presence and function of multiple type I signal peptidase isoforms was investigated. In addition to type I signal peptidases SipS(ba) [Meijer, W.J.J., de Jong, A., Bea, G., Wisman, A., Tjalsma, H., Venema, G., Bron, S. & van Dijl, J.M. (1995) Mol. Microbiol. 17, 621-631] and SipT(ba) [Hoang, V. & Hofemeister, J. (1995) Biochim. Biophys. Acta 1269, 64-68] which were previously identified, here we present evidence for two other Sip-like genes in B. amyloliquefaciens. Same map positions as well as sequence motifs verified that these genes encode homologues of Bacillus subtilis SipV and SipW. SipU-encoding DNA was not found in B. amyloliquefaciens. SipW-encoding DNA was also found for other Bacillus strains representing different phylogenetic groups, but not for Bacillus stearothermophilus and Thermoactinomyces vulgaris. The absence of these genes, however, could have been overlooked due to sequence diversity. Sequence alignments of 23 known Sip-like proteins from Bacillus origin indicated further branching of the P-group signal peptidases into clusters represented by B. subtilis SipV, SipS-SipT-SipU and B. anthracis Sip3-Sip5 proteins, respectively. Each B. amyloliquefaciens sip(ba) gene was expressed in an Escherichia coli LepBts mutant and tested for genetic complementation of the temperature sensitive (TS) phenotype as well as pre-OmpA processing. Although SipS(ba) as well as SipT(ba) efficiently restored processing of pre-OmpA in E. coli, only SipS(ba) supported growth at TS conditions, indicating functional diversity. Changed properties of the sip(ba) gene disruption mutants, including cell autolysis, motility, sporulation, and nuclease activities, seemed to correlate with specificities and/or localization of B. amyloliquefaciens SipS, SipT and SipV isoforms.

Bacillus↗
PubMed1997

Peptidases that degrade gonadotropin-releasing hormone: influence on LH secretion in the ewe.

Several peptidases have been postulated to degrade the hypothalamic peptide gonadotropin-releasing hormone (GnRH), but it is not known if such enzymes contribute significantly to the delivery of GnRH to the pituitary in vivo. Furthermore, the activity of GnRH-inactivating peptidases may vary in different reproductive states, such as across the estrous cycle. In the present study, specific fluorescent substrates were used to measure the activity of the two major GnRH-degrading enzymes, prolyl endopeptidase (PEP) and endopeptidase 3.4.24.15 (EP 24.15), in soluble extracts of the median eminentes (ME) of ewes during different phases of the estrous cycle. Levels of EP 24.15 and PEP activity in the ME did not vary significantly across the cycle, although PEP activity was lowest at the time of the preovulatory luteinizing hormone (LH) surge. However, a statistically significant decline in PEP activity (18%, P = 0.02) was observed in the ME of OVX ewes in which a surge was induced by estrogen when compared to oil-treated OVX controls, suggesting a possible negative regulation of PEP activity by this steroid. The effect of intracerebroventricular (i.c.v.) infusion of several peptidase inhibitors on the pulsatile release of LH in the conscious OVX ewe was also examined. No consistent changes in the pattern of LH release were observed with i.c.v. infusion of the EP 24.15 inhibitor N-[1(R,S)-carboxy-3-phenylpropyl]-Ala-Ala-Tyr-p-aminobenzoate (cFP-AAY-pAB) or the angiotensin-converting enzyme (ACE) inhibitor captopril. Similarly, administration of the prolyl endopeptidase inhibitor bacitracin, or a more specific inhibitor of this enzyme, Z-Proprolinal (ZPP), did not alter LH release patterns. The results did not demonstrate a major role for changes in the activity of EP 24.15, PEP, or ACE in altering the pattern of GnRH secretion, but a minor reduction in PEP levels may occur at the time of the estrogen-induced LH surge.

Angiotensin-Converting Enzyme Inhibitors↗
PubMed2003

Cleavage of preflagellins by an aspartic acid signal peptidase is essential for flagellation in the archaeon Methanococcus voltae.

The differences between archaeal and bacterial flagella are becoming more apparent as research on the archaeal structure progresses. One crucial difference is the presence of a leader peptide on archaeal preflagellins, which is removed from the flagellin prior to its incorporation into the flagellar filament. The enzyme responsible for the removal of the flagellin leader peptide was identified as FlaK. FlaK of Methanococcus voltae retains its preflagellin peptidase activity when expressed in Escherichia coli and used in an in vitro assay. Homologous recombination of an integration vector into the chromosomal copy of flaK resulted in a non-motile, non-flagellated phenotype. The flagellins of the mutant had larger molecular weights than their wild-type counterparts, as expected if they retained their 11- to 12-amino-acid leader peptide. Membranes of the flaK mutant were unable to process preflagellin in the in vitro assay. Site-directed mutagenesis demonstrated that two aspartic acid residues conserved with ones in type IV prepilin peptidases were necessary for proper recognition or processing of the preflagellin. As bacterial flagellins lack a leader peptide and a peptidase is not required for export and assembly, the requirement for FlaK further emphasizes the similarity archaeal flagella have with type IV pili, rather than with bacterial flagella.

Amino Acid Sequence↗
PubMed1976

[Collagen peptidase in Crohn's disease].

Collagen-peptidase activity in serum was increased in 54 of 71 cases of Crohn's disease. This increase was highly significant (P less than 0.001) compared with the results in a control group (24 cases). Immunosuppressive drugs (corticosteroids, azathioprine) decreased collagen-peptidase activity in the patients with Crohn's disease. On average it was higher than that of patients with sarcoidosis. Further tests are required to establish whether collagen-peptidase activity is of diagnostic value in Crohn's disease.

Adrenal Cortex Hormones↗
PubMed2002

Endoproteolysis by isolated membrane peptidases reveal metabolic stability of glucagon-like peptide-1 analogs, exendins-3 and -4.

These in vitro studies aimed to characterize the pattern and the kinetics of endoproteolysis of the insulinotropic hormone glucagon-like peptide-1 (GLP-1) and related peptides by native ectopeptidases. Peptides were incubated with isolated rat or pig kidney brush-border microvilli membranes, which are a rich source of the ectopeptidases that are responsible for the post-secretory metabolism of peptide hormones. The proteolytic products were separated by reversed-phase HPLC column chromatography and characterised by molecular mass and primary structure. The relative importance of specific peptidases was established by measuring the effects of specific peptidase inhibitors on the kinetics of proteolysis. Dipeptidyl-peptidase-IV was found to be rate-limiting in the endoproteolysis of GLP-1. GLP-1 homologs, exendins-3 and -4, exhibited exceptional stability in the presence of isolated kidney microvilli membranes. Our finding that exendin-4 is several orders of magnitude more stable than GLP-1 and Ser-8-GLP-1 is especially noteworthy given this peptide's widely reported insulinotropic potency.

Animals↗
PubMed2004

The molecular structure and catalytic mechanism of a novel carboxyl peptidase from Scytalidium lignicolum.

The molecular structure of the pepstatin-insensitive carboxyl peptidase from Scytalidium lignicolum, formerly known as scytalidopepsin B, was solved by multiple isomorphous replacement phasing methods and refined to an R factor of 0.230 (R(free) = 0.246) at 2.1-A resolution. In addition to the structure of the unbound peptidase, the structure of a product complex of cleaved angiotensin II bound in the active site of the enzyme was also determined. We propose the name scytalidocarboxyl peptidase B (SCP-B) for this enzyme. On the basis of conserved, catalytic residues identified at the active site, we suggest the name Eqolisin for the enzyme family. The previously uninvestigated SCP-B fold is that of a beta-sandwich; each sheet has seven antiparallel strands. A tripeptide product, Ala-Ile-His, bound in the active site of SCP-B has allowed for identification of the catalytic residues and the residues in subsites S1, S2, and S3, which are important for substrate binding. The most likely hydrolytic mechanism involves nucleophilic attack of a general base (Glu-136)-activated water (OH(-)) on the si-face of the scissile peptide carbonylcarbon atom to form a tetrahedral intermediate. Electrophilic assistance and oxyanion stabilization is provided by the side-chain amide of Gln-53. Protonation of the leaving-group nitrogen is accomplished by the general acid function of the protonated carboxyl group of Glu-136.

Amino Acid Sequence↗
PubMed1988

The cytoplasmic domain of Escherichia coli leader peptidase is a "translocation poison" sequence.

Leader peptidase is an integral, transmembrane protein of the plasma membrane of Escherichia coli. Its membrane assembly requires its internal, uncleaved signal sequence, its large periplasmic carboxyl-terminal region, and an apolar domain that is known as a "hydrophobic helper." We now show that the polar cytoplasmic domain of leader peptidase is a unique membrane assembly element, which we term a "translocation poison" sequence. This sequence is defined by its ability to block the action of a signal sequence that either precedes or follows it. To our knowledge, this is the first entirely polar topogenic element. Deletion analysis shows that the role of the leader peptidase hydrophobic helper sequence in its membrane assembly is to overcome the block to assembly caused by the poison sequence.

Cell Membrane↗
PubMed1994

Peptidase activities of proteasomes are differentially regulated by the major histocompatibility complex-encoded genes for LMP2 and LMP7.

Recent studies have implicated proteasomes in the generation of the antigenic peptides that are presented on major histocompatibility complex class I molecules to T lymphocytes. Interferon gamma modifies the subunit composition of proteasomes and causes changes in their peptidase activities that should favor the production of peptides with hydrophobic or basic carboxyl termini (i.e., the types found on major histocompatibility complex class I molecules). It has been proposed that these changes in peptidase activity are due to incorporation into proteasomes of the major histocompatibility complex-encoded subunits LMP2 and -7, which are induced by interferon gamma. Here we show by gene transfection into lymphoblasts or HeLa cells that LMP7 increases the capacity (Vmax) of 20S and 26S proteasomes to cleave peptides after hydrophobic and basic residues without affecting hydrolysis after acidic residues. These changes depended on the amount of LMP7 subunits incorporated into proteasomes. Transfection of LMP2 reduced cleavage of peptides after acidic residues, increased hydrolysis after basic residues, and did not affect the hydrophobic activity. Since the activity of the total proteasome population changed after incorporation of only small amounts of LMP2 or -7, these subunits must cause major alterations in peptidase activity. Thus, their expression can account for the changes in proteasome activity induced by inteferon gamma, and these findings lend further support to the proposed roles of LMPs in altering the nature of the peptides generated for antigen presentation.

Amino Acid Sequence↗
PubMed1997

Bacillus subtilis contains four closely related type I signal peptidases with overlapping substrate specificities. Constitutive and temporally controlled expression of different sip genes.

Most biological membranes contain one or two type I signal peptidases for the removal of signal peptides from secretory precursor proteins. In this respect, the Gram-positive bacterium Bacillus subtilis seems to be exceptional, because it contains at least four chromosomally-encoded type I signal peptidases, denoted SipS, SipT, SipU, and SipV. Here, we report the identification of the sipT and sipV genes, and the functional characterization of SipT, SipU, and SipV. The four signal peptidases have similar substrate specificities, as they can all process the same beta-lactamase precursor. Nevertheless, they seem to prefer different pre-proteins, as indicated by studies on the processing of the pre-alpha-amylase of Bacillus amyloliquefaciens in strains lacking SipS, SipT, SipU, or SipV. The sipU and sipV genes are constitutively transcribed at a low level, suggesting that they are required for processing of (pre-)proteins secreted during all growth phases. In contrast, the transcription of sipS and sipT is temporally controlled, in concert with the expression of the genes for most secretory proteins, which suggests that SipS and SipT serve to increase the secretory capacity of B. subtilis. Taken together, our findings suggest that SipS, SipT, SipU, and SipV serve different functions during the exponential and post-exponential growth phase of B. subtilis.

Amino Acid Sequence↗
PubMed1997

The cysteine-peptidase bleomycin hydrolase is a member of the galactose regulon in yeast.

Bleomycin hydrolase is a cysteine peptidase discovered through its ability to detoxify the anti-cancer glycopeptide, bleomycin. Although found in all tissues in mammals and in both eukaryotes and prokaryotes, the normal cellular function of this peptidase is not known. We had previously reported the purification of bleomycin hydrolase from yeast based on its unexpected ability to bind DNA. Recently we collaborated in solving the crystal structure of this protein, revealing a hexameric ring organization. We now report that the molecular characterization of the gene encoding yeast bleomycin hydrolase is also surprising. The transcription of the gene is regulated by galactose. Furthermore, this regulation is conveyed by a binding site for the Gal4 regulatory protein in its promoter, prompting the designation of this gene as GAL6. Gal6p also appears to have a negative effect on the GAL system as a deletion of the gene leads to a 2-5-fold higher expression of the GAL1, GAL2, GAL7, and MEL1 genes. The GAL6 deletion does not affect the expression of another inducible gene, HSP26. Neither the peptidase nor the nucleic acid binding activity of Gal6p as assayed is apparently required to convey this regulation, implying yet another function for this new member of the GAL regulon.

Base Sequence↗
PubMed1999

Different mechanisms for thermal inactivation of Bacillus subtilis signal peptidase mutants.

The type I signal peptidase SipS of Bacillus subtilis is of major importance for the processing of secretory precursor proteins. In the present studies, we have investigated possible mechanisms of thermal inactivation of five temperature-sensitive SipS mutants. The results demonstrate that two of these mutants, L74A and Y81A, are structurally stable but strongly impaired in catalytic activity at 48 degrees C, showing the (unprecedented) involvement of the conserved leucine 74 and tyrosine 81 residues in the catalytic reaction of type I signal peptidases. This conclusion is supported by the crystal structure of the homologous signal peptidase of Escherichia coli (Paetzel, M., Dalbey, R. E., and Strynadka, N. C. J. (1998) Nature 396, 186-190). In contrast, the SipS mutant proteins R84A, R84H, and D146A were inactivated by proteolytic degradation, indicating that the conserved arginine 84 and aspartic acid 146 residues are required to obtain a protease-resistant conformation. The cell wall-bound protease WprA was shown to be involved in the degradation of SipS D146A, which is in accord with the fact that SipS has a large extracytoplasmic domain. As WprA was not involved in the degradation of the SipS mutant proteins R84A and R84H, we conclude that multiple proteases are responsible for the thermal inactivation of temperature-sensitive SipS mutants.

Bacillus subtilis↗
PubMed1999

Signal peptide peptidase- and ClpP-like proteins of Bacillus subtilis required for efficient translocation and processing of secretory proteins.

Signal peptides direct the export of secretory proteins from the cytoplasm. After processing by signal peptidase, they are degraded in the membrane and cytoplasm. The resulting fragments can have signaling functions. These observations suggest important roles for signal peptide peptidases. The present studies show that the Gram-positive eubacterium Bacillus subtilis contains two genes for proteins, denoted SppA and TepA, with similarity to the signal peptide peptidase A of Escherichia coli. Notably, TepA also shows similarity to ClpP proteases. SppA of B. subtilis was only required for efficient processing of pre-proteins under conditions of hyper-secretion. In contrast, TepA depletion had a strong effect on pre-protein translocation across the membrane and subsequent processing, not only under conditions of hyper-secretion. Unlike SppA, which is a typical membrane protein, TepA appears to have a cytosolic localization, which is consistent with the observation that TepA is involved in early stages of the secretion process. Our observations demonstrate that SppA and TepA have a role in protein secretion in B. subtilis. Based on their similarity to known proteases, it seems likely that SppA and TepA are specifically required for the degradation of proteins or (signal) peptides that are inhibitory to protein translocation.

Adenosine Triphosphatases↗