Gene Cloning and Characterization of PepC, a Cysteine Aminopeptidase from Streptococcus thermophilus, with sequence Similarity to the Eucaryotic Bleomycin Hydrolase (original) (raw)
Related papers
St-PepA, a Streptococcus thermophilus aminopeptidase with high specificity for acidic residues
Microbiology, 1995
The proteolytic system of lactic acid bacteria has been extensively studied over the past 10 years and peptidases from lactococci are now well known. The situation is, however, different for S t~~t o~o c c u s thennophilus from which only a few peptidases have been purified and characterized. The present work was conducted to characterize an aminopeptidase of S. thermophilus CNRZ 302, called St-Pep& with high specificity for acidic amino acids. St-PepA was purified by a three-step procedure from a spheroblast extract of S. thermophilus CNRZ 302. Its molecular mass was estimated to be 360 kDa by gel filtration and 45 kDa by SDS-PAGE, indicating that it had an octameric structure. Its activity against aspartate-p-nitroanilide was maximal at pH 8.5 and 62 "C and highly enhanced by Znz+, Coz+ and Mgz+. St-PepA was inhibited by metal-chelating reagents and, to a lesser extent, by agents modifying sulfhydryl groups. It showed an activity towards p-nitroanilide derivatives, diand tripeptides, and larger peptides such as fragment 43-58 of a,,-casein. It had a high substrate specificity towards N-terminal acidic amino acid residues but it could also release serine and malic acid, the a-hydroxy acid homologue of aspartic acid. Kinetic studies revealed that the affinity of St-PepA was more than l&fold higher for aspartic acid-p-nitroanilide (K, = 0.42 mM) than for glutamic acid-pnitroanilide (Km = 7 6 5 mM) with a similar V-for both substrates [about 40 pmol min-l (mg enzyme)-']. St-PepA is heat stable, with a maximal loss of activity of 15% after incubation for 120 min at 50 "C and 60 "C. It is likely to be involved in the nitrogen metabolism of S. thermophilus and in the development of the organoleptic characteristics of cheese and yoghurt.
1991
The X-prolyl-dipeptidyl aminopeptidase from Streptococcus macedonicus ACA-DC 191 was purified by anion exchange and hydrophobic interaction chromatography. A single band of a molecular mass of about 84 000 g•mol-1 appeared in SDS-PAGE; by gel filtration it was shown that the native enzyme was dimeric. The enzyme showed optimum activity on glycyl-prolyl-4-nitroanilide at pH 7.0, with a K M = 0.42 mmol•L-1 and a V max = 12.8 µmol•mg-1 •min-1. It was active over a temperature range of 10-60 o C. Over 60 o C, the enzyme activity declined rapidly. The peptidase was completely inactivated by PMSF, DTNB and Cu 2+ , while metal chelators had no effect on enzyme activity. By using the PCR technique with synthetic primers, the pepX gene was amplified, cloned and sequenced. This 2 289 nucleotide gene encodes a protein of 763 amino acids with a molecular mass of 86 866 g•mol-1. The deduced amino acid sequence analysis of the pepX gene shows a high identity with PepX enzymes from other lactic acid bacteria and contains a motif around the active site serine (G-K-S-Y-L-G) that is well conserved among the PepX enzymes. Streptococcus macedonicus / X-prolyl-dipeptidyl aminopeptidase / PepX purification / enzyme characterization / cloning Résumé-Purification et caractérisation de la X-prolyl-dipeptidyl aminopeptidase (PepX) de Streptococcus macedonicus et clonage du gène pepX. Une X-prolyl-dipeptidyl aminopeptidase extraite de Streptococcus macedonicus ACA-DC 191 a été purifiée par chromatographie échangeuse d'anions et chromatographie d'intéractions hydrophobes. Une seule bande de masse moléculaire de 84 000 g•mol-1 a été detectée sur SDS-PAGE ; par chromatographie d'exclusion de taille il a été démontré que l'enzyme native était dimérique. L'enzyme a une activité maximale sur le substrat glycylprolyl-4-nitroanilide à pH 7,0, avec K M = 0,42 mmol•L-1 et V max = 12,8 µmol•mg-1 •min-1. Elle est active dans une gamme de températures de 10-60 o C. Au-dessus de 60 o C, l'activité enzymatique 657
Catalytic properties of the cysteine aminopeptidase PepC, a bacterial bleomycin hydrolase
Biochimica et Biophysica Acta (BBA) - Protein Structure and Molecular Enzymology, 1998
PepC is a cytoplasmic thiol aminopeptidase widely conserved among lactic acid bacteria. PepC from Lactococcus lactis shares 35-38% identity with aminopeptidases of eukaryotic origins: the yeast and mammalian bleomycin hydrolases Ž. BLMase. In this work we investigated the hydrolytic activity of PepC towards various substrates: bleomycin A2, Ž. aminoacyl-p-nitroanilides pNA and peptides. First, we found the bleomycin hydrolase activity of lactococcal PepC and measured similar kinetics parameters to those reported for the mammalian BLMase. Second, the results obtained on aminoacyl-pNA confirmed the capacity of the enzyme to release a broad range of amino acids and the pH activity profile suggests the presence of an ionic interaction between the enzyme and the free a-amino group of the substrate. Third, the aminopeptidase activity measured on peptide substrates revealed that PepC possesses an extended binding site which interacts with the peptidic backbone of the substrate. The hydrolytic efficiency is highly dependent on the length of the peptide, optimal for tetrapeptides and further enhanced by the presence of hydrophobic residues in the P X positions of the substrate. These enzymatic properties are of importance for the design of specific inhibitors and the biological function of the bleomycin hydrolases. q 1998 Elsevier Science B.V.
European Journal of Biochemistry, 1999
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 <45 kDa with optimal activity in the range pH 7.5±8.5 and at 55 8C 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.
FEBS Letters, 1992
The nucleotide scqucncc of the pci~'I,N gene from toc/orocc.rt.s Iurrb erlcoding a zinc-metallo aminopeptidase has been determined. The open reading frame of 2,538 base pairs encodes a protein with a calculated ilf, of 95,368, which aprccs with the apparent M,of95,000 of the gene product which was identified by polyclonal antibodies raised against the purified aminopcptidasc. The amino acid sequence of the aminopeptidase of L. fuctis wss found to be similar to the corresponding cnzymcs of human, rat and mouse, with host 30% of the residues identical. Also. a highly conserved arca was identified which has similarity with the uctivc site of thcrmolysin. A zinc-binding site, as well as the catalytic site for RpN, is predicted to lie within this conscrvcd stretch, Putative promoter regions upstream of PcpN were confirmed by primer extension analysis. Aminopctxidasc N; Lacrococar,r lurris; Mammalian _ Correspondcc uddress: WN:. Koninys, Dcpartmcnt of Microbiology. University uf Groningen. Kcrklaon 30.9751 NN Harcn, The Netherlands. Fax: (3 I) (50) 635 205.
Journal of Applied Microbiology, 2000
To investigate the effect of an absence of aminopeptidase PepS on the growth of Streptococcus thermophilus on different media and at different temperatures. Using gene interruption, a negative mutant of the Strep. thermophilus CNRZ385 strain was constructed for the aminopeptidase PepS (strain DeltapepS). Checks were first of all made using biochemical assays that the DeltapepS strain lacks the peptide hydrolase activity of aminopeptidase PepS. It was demonstrated that the absence of the aminopeptidase PepS exerted a negative effect on growth whatever the culture medium (M17, chemically defined medium, milk). The role of aminopeptidase PepS in growth was enhanced at a high temperature (45 degrees C vs 37 degrees C). The DeltapepS strain was more resistant to lysozyme than the wild-type strain. We were able to demonstrate that aminopeptidase PepS probably plays a pleiotropic role through its involvement in growth via nitrogen nutrition, as well as via other cellular functions/metabolisms (such as peptidoglycane metabolism). This study constitutes the first report on the role of a member of the M29 MEROPS family of metallopeptidases (http://merops.sanger.ac.uk/).
Journal of Applied Microbiology, 1997
F . R UL A ND V. M ON NE T . 1997. Streptococcus thermophilus is widely used in the dairy industry but little is known about its peptidase system. The aim of this study was to determine the biochemical and genetic characteristics of this system, and to compare it to the well known system of Lactococcus lactis. We separated the intracellular proteins of Strep. thermophilus CNRZ 302 and L. lactis NCDO 763 by ion-exchange chromatography and we detected the activity of the different types of peptidases. In both L. lactis and Strep. thermophilus strains, we showed 13 different peptidase activities with biochemical homologies between both species. Streptococcus thermophilus also possessed two peptidases which we did not find in L. lactis: an aminopeptidase and an oligopeptidase. We performed Southern blot experiments and among the eight peptidase genes tested, only the genes encoding the general aminopeptidases, pepC and pepN, were homologous between the L. lactis and Strep. thermophilus strains. Besides biochemical and genetic similarities, the peptidase systems of Strep. thermophilus and L. lactis thus differed by the presence of additional peptidases in Strep. thermophilus.
Fems Microbiology Letters, 1994
Abstract An aminopeptidase N (pepN) gene was detected by DNA hybridization from an industrially important Lactobacillus helveticus strain using part of the L. helveticus CNRZ32 pepN gene as the probe. One of five hybridization positive clones was characterized in more detail. A subcloned 3.7 kb fragment, positive in hybridization and encoding aminopeptidase activity, was sequenced and analyzed. Only one open reading frame (ORF) of 2532 base pairs with a coding capacity for a 95.9 kDa protein could be found. The deduced amino acid sequence of the 95.9 kDa protein showed homology to PepN proteins from other lactic acid bacteria and carried the conserved catalytic and zinc binding sites of the neutral zinc metallo-peptidase family confirming the identity of the pepN gene. A 2.75 kb transcript and two transcription start sites were identified with mRNA analyses. Expression of pepN in L. helveticus, studied as the function of growth, revealed a high level of pepN transcripts throughout the growth, in contrast to the steady state levels of other peptidase mRNAs from L. helveticus analyzed in our laboratory.
European Food Research and Technology, 2000
The aminopeptidase C (PepC) of Lactobacillus helveticus CNRZ32 was purified by anion exchange chromatography from cell free extracts of an E. coli DH5α clone overexpressing the Lactobacillus aminopeptidase. PepC was found to have a tetrameric structure in its native form with subunits of 50 kDa each, a pH optimum of 6.5 and maximum activity at 45 °C. Sulfhydryl-blocking reagents inhibited the enzyme activity whereas reducing or metal chelating reagents had an activating effect on the PepC activity. The PepC hydrolyzed a wide range of p-nitroaniline derivatives, dipeptides and several tripeptides which contained basic amino acids (Arg, Lys), Pro residues, or cheese flavour precursor amino acids (Met, Leu, Phe) at the N-terminal position. The substrate specificity and residual activity of PepC from several lactic acid bacteria, including the PepC described above, were compared at conditions of pH and NaCl present in cheese.
European Journal of Biochemistry, 1994
An aminopeptidase C gene (pepC) was detected by nucleic acid hybridization from an industrially important Lactobacillus helveticus strain. Three hybridization positive clones were isolated from a gene library of this L. helveticus strain, and one of them was characterized in more detail. Deletion mapping localized the hybridization positivity into a 2.8-kb fragment, which also encoded aminopeptidase activity. This fragment was sequenced and two open reading frames (ORF1 and 2) of 1347 and 840 base pairs were identified. The ORFl was preceded by a typical prokaryotic promoter region, and an inverted repeat structure with AG of -49.0 kJ mol-' was found downstream of the coding region. The deduced amino acid sequence of ORF1, with an encoding capacity for a 51.4-kDa protein, was shown to share 48.3% and 98.0% identities with the PepC proteins from Lactococcus lactis and L. helveticus CNRZ32, respectively, thus confirming that ORFl codes for an aminopeptidase C. mRNA size analyses revealed 1.7-kb and 2.7-kb transcripts in Northern blot with the pepC-specific probe. A further analysis with the pepCand ORF2-specific probes showed that downstream ORF2 is co-transcribed with the pepC gene at the exponential phase of growth whereas, at the stationary growth phase, transcripts derived from the pepC promoter were below the detection limit, and the ORF2 was expressed by its own promoter. The 5' end mapping of the pepC