Gewinnung und Charakterisierung von Prolidase aus Escherichia coli B,II. Charakterisierung,Reinigung und Trägerfixierung des Enzyms

Prolidase, a specific exopeptidase, is isolated from Escherichia coli B. The enzyme being present in the raw extract is purified and enriched by fractionated ammonium sulfate precipitation, ion exchange chromatography on DEAE-Sephadex A 50 as well as by gel filtration on Sepharose 4 B. Total yield of prolidase amounts to 19% with a 67fold enrichment Substrate specifity of the enzyme mainly corresponds to that of the animal prolidase. It is able to hydrolize the imido linkage at the N-terminal end of prolin in the case of di- and tripeptides. The temperature optimum of prolidase from E. coli B is 37 °C, the pH-optimum from pH 7.6 to 9.0. Storage stability at pH 8.6 and a temperature of 4 °C is optimal. The enzyme is only active in presence of Mn²⁺-ions. This metal cannot be replaced by Mg^2-- or Zn²⁺-ions A high enzyme activity and storage stability in presence of Mn²⁺-ions can be reached by immobilization of the prolidase, by covalent binding on Sepharose 6 B, adsorption on DEAE-Sephadex as well as by combination with glutar dialdehyde on DEAE-Sephadex.     

Phosphorylation of prolidase increases the enzyme activity

Prolidase [EC 3.4.13.9] is a ubiquitously distributed imidodipeptidase that catalyzes the hydrolysis of C-terminal proline-containing dipeptides. The enzyme plays an important role in the recycling of proline for collagen synthesis and cell growth. Although, the increase in the enzyme activity is correlated with increased rate of collagen turnover, the mechanism by which prolidase is regulated remain largely unknown. In the present study we found that phosphorylation of fibroblast's prolidase may be an underlying mechanism for up regulation of the enzyme activity. Supporting evidence comes from the following observations: (1) immunoprecipitated prolidase was detected as a phosphotyrosine protein as shown by western immunoblot analysis, (2) tyrosine kinase inhibitor – erbstatin induced (in a dose dependent manner) a decrease in prolidase activity in cultured human skin fibroblasts, (3) anti-phosphotyrosine antibody reduced and phosphotyrosine phosphatase 1B antibody (anti-PTP 1B) increased (in a dose dependent manner) the prolidase activity in extract of fibroblast's homogenate, (4) decrease in prolidase activity from collagenase treated or serum starved fibroblasts can be partially prevented by incubating fibroblast's homogenate extract with anti-PTP 1B antibody. These results provide evidence that prolidase is phosphotyrosine enzyme and suggest that the activity of prolidase may be up regulated by the enzyme phosphorylation.     

Inhibition of prolidase activity by nickel causes decreased growth of proline auxotrophic CHO cells

Occupational exposure to nickel has been epidemiologically linked to increased cancer risk in the respiratory tract. Nickel-induced cell transformation is associated with both genotoxic and epigenetic mechanisms that are poorly understood. Prolidase [E.C.3.4.13.9] is a cytosolic Mn(II)-activated metalloproteinase that specifically hydrolyzes imidodipeptides with C-terminal proline or hydroxyproline and plays an important role in the recycling of proline for protein synthesis and cell growth. Prolidase also provides free proline as substrate for proline oxidase, whose gene is activated by p53 during apoptosis. The inhibition of prolidase activity by nickel has not yet been studied. We first showed that Ni(II) chloride specifically inhibited prolidase activity in CHO-K1 cells in situ. This interpretation was possible because CHO-K1 cells are proline auxotrophs requiring added free proline or proline released from added Gly-Pro by prolidase. In a dose-dependent fashion, Ni(II) inhibited growth on Gly-Pro but did not inhibit growth on proline, thereby showing inhibition of prolidase in situ in the absence of nonspecific toxicity. Studies using cell-free extracts showed that Ni(II) inhibited prolidase activity when present during prolidase activation with Mn(II) or during incubation with Gly-Pro. In kinetic studies, we found that Ni(II) inhibition of prolidase varied with respect to Mn(II) concentration. Analysis of these data suggested that increasing concentrations of Mn(II) stabilized the enzyme protein against Ni(II) inhibition. Because prolidase is an important enzyme in collagen metabolism, inhibition of the enzyme activity by nickel could alter the metabolism of collagen and other matrix proteins, and thereby alter cell-matrix and cell-cell interactions involved in gene expression, genomic stability, cellular differentiation, and cell proliferation.     

Improved production of holotoxin Stx2 with biological activities by using a single-promoter vector and an auto-induction expression system

The entire stx2 region from Escherichia coli O157:H7, containing two open reading frames (stx2a and stx2b), was cloned into pET-32a with a single promoter, and transformed into E. coli BL21 (DE3) pLysS. We used two methods of IPTG induction using LB medium and auto-induction using ZYM-5052 medium to express recombinant Shiga toxin 2 (rStx2). rStx2 was expressed in the E. coli periplasm in a completely soluble, biologically active form. The final yield of purified rStx2 from each liter of culture in LB medium and ZYM-5052 medium was approximately 2.3 mg and 3.5 mg, respectively. The highest amount of rStx2 accounted for 27.8% of total bacteria protein in ZYM-5052 medium. rStx2A and rStx2B isolated from rStx2 by electroelution were, respectively, identified by N-terminal protein sequencing. Signal peptides with the sequence MKCILFKWVLCLLLGFSSVSYS and MKKMFMAVLFALASVNAMA were identified at the N terminus of rStx2A and rStx2B, respectively. Our rStx2 possessed Vero cell CD₅₀ value about 500 pg and LD₅₀ value approximately 6 ng. rStx2 can be substitute for natural toxin Stx2, which can be used for animal models, drug screening, vaccine research, and so on.     

Biosynthesis of polyribonucleotide phosphorylase and polyribonucleotides by E. Coli

The biochemical functions of Polyribonucleotide phosphorylase (PNP-ase; EC 2.7.7.8) has been studied The present work was aimed at studying the interrelation between PNP-ase biosynthesis and RNA content of E. coli cells under various conditions of bacterial growth, obtaining the biomass of E. coli with high PNP-ase activity as well as to study the possibility of secondary cultivation of E. coli cells for obtaining polyribonucleotides from nucleoside-5′-diphosphates The spcific PNP-ase activity increases at secondary cultivation, when the medium contains ribonucleoside-5′-diphosphates. Besides one may observe an extracellular polycondensation of nucleoside-5′-diphosphates It may be presumed that the PNP-ase is a multifunctional enzyme whose biological role may be confined to regulating the energetic processes connected with the metabolism of glucose, nucleosidephosphates and orthophosphate in the cells of E. coli.     

Additional Volatile Compounds Produced by Pyrolysis of Sulfur-containing Amino Acids

Bacillus circulans WL-12, a yeast and fungal cell wall lytic bacterium, secretes a variety of polysaccharide degrading enzymes into the culture medium. When β-1,3-glucanase was induced with pachyman, a β-1,3-glucose polymer obtained from the tree fungus Poria cocus Wolf, six distinct active molecules of the enzyme with different molecular weights were detected in the culture supernatant of this bacterium. Molecular cloning of one of the β,3-gIucanase genes into E. coli was achieved by transforming E. coli HB101 cells with recombinant plasmids composed of chromosomal DNA fragments prepared from B. circulans WL-12 and the plasmid vector pUC 19. A recombinant plasmid containing 4.4 kb of inserted DNA in the Pst I site of pUC 19, designated as pNT003, conferred the ability to degrade pachyman on E. coli cells. The presence of pNT003 was harmful for E. coli cells and caused cell lysis, especially at higher temperatures of cultivation. β,3-Glucanase activity detected in E. coli was mainly recovered in the periplasmic fraction when cell lysis did not occur. SDS-PAGE analysis revealed that the periplasmic fraction contained four active molecules of β-1,3-glucanase which corresponded to four of the six active molecules produced by B. circulans WL-12.     

Prolidase activity in fibroblasts is regulated by interaction of extracellular matrix with cell surface integrin receptors

Prolidase (EC 3.4.13.9) is a ubiquitously distributed imidodipeptidase that catalyzes the hydrolysis of C-terminal proline or hydroxyproline containing dipeptides. The enzyme plays an important role in the recycling of proline for collagen synthesis and cell growth. An increase in enzyme activity is correlated with increased rates of collagen turnover indicative of extracellular matrix (ECM) remodeling, but the mechanism linking prolidase activity and ECM is poorly understood. Thus, the effect of ECM-cell interaction on intracellular prolidase activity is of special interest. In cultured human skin fibroblasts, the interaction with ECM and, more specifically, type I collagen mediated by the β₁ integrin receptor regulates cellular prolidase activity. Supporting evidence comes from the following observations: 1) in sparse cells with a low amount of ECM collagen or in confluent cells in which ECM collagen was removed by collagenase (but not by trypsin or elastase) treatment, prolidase activity was decreased; 2) this effect was reversed by the addition of type I collagen or β₁ integrin antibody (agonist for β₁ integrin receptor); 3) sparse cells (with typically low prolidase activity) showed increased prolidase activity when grown on plates coated with type I collagen or on type IV collagen and laminin, constituents of basement membrane; 4) the relative differences in prolidase activity due to collagenase treatment and subsequent recovery of the activity by β₁ integrin antibody or type I collagen treatment were accompanied by parallel differences in the amount of the enzyme protein recovered from these cells, as shown by Western immunoblot analysis. Thus, we conclude that prolidase activity responded to ECM metabolism (tissue remodeling) through signals mediated by the integrin receptor. J. Cell. Biochem. 67:166–175, 1997. Published 1997 Wiley-Liss, Inc.     

Characterization of two proline dipeptidases (prolidases) from the hyperthermophilic archaeon Pyrococcus horikoshii

Prolidases hydrolyze the unique bond between X-Pro dipeptides and can also cleave the P–F and P–O bonds found in organophosphorus compounds, including the nerve agents, soman and sarin. The advantages of using hyperthermophilic enzymes in biodetoxification strategies are based on their enzyme stability and efficiency. Therefore, it is advantageous to examine new thermostable prolidases for potential use in biotechnological applications. Two thermostable prolidase homologs, PH1149 and PH0974, were identified in the genome of Pyrococcus horikoshii based on their sequences having conserved metal binding and catalytic amino acid residues that are present in other known prolidases, such as the previously characterized Pyrococcus furiosus prolidase. These P. horikoshii prolidases were expressed recombinantly in the Escherichia coli strain BL21 (λDE3), and both were shown to function as proline dipeptidases. Biochemical characterization of these prolidases shows they have higher catalytic activities over a broader pH range, higher affinity for metal and are more stable compared to P. furiosus prolidase. This study has important implications for the potential use of these enzymes in biotechnological applications and provides further information on the functional traits of hyperthermophilic proteins, specifically metalloenzymes.     

Caffeine-death in Escherichia coli

Summary Growth of a culture of E. coli strain B or 15 in medium containing caffeine resulted in the accumulation of inviable cells in the population. A caffeine concentration of 8 mM caused the death of between 30% and 50% of the cells in 12 independent populations grown for 15 generations or more. The thymine dimer excision-defective strains Bs-1, Bs-8 and Bs-12 and the exr ^– mutant Bs-2 were resistant to this lethal effect. The reckless, hcr ⁺ mutant Bs-11 was more sensitive than the parental B strain. Although 100mM caffeine did not impair DNA synthesis in vitro, concentrations of the drug 8 mM caused a significant decline in DNA synthesis in vivo in E. coli B cells. From the fit of an experimental growth curve to an algebraic model of growth in which a proportion of cells are inactivated at each replication it is suggested that caffeine does not affect the replication rate of the viable cells. The observed impairment of DNA synthesis in vivo is equated with this cell death (caffeine-death). For E. coli 15 or B, 8 mM caffeine induced caffeine-death at a rate of 18% per cell generation. Caffeine-resistant mutants of E. coli B and E. coli 15 were isolated. Of those studied in detail a substantial proportion proved to be U.V. and X-ray sensitive and excision-defective. Others were more U.V. and X-ray resistant than strain B. Yet another class proved highly unstable. A chromosome breakage model of caffeine-death implicating enzymes of the excision-repair process is discussed.     

Features of dnaK operon genes of the obligate thermophile Bacillus thermoglucosidasius KP1006

The dnaK gene was cloned from the obligate thermophile Bacillus thermoglucosidasius KP1006, together with the grpE and dnaJ genes in the same operon. The dnaK, grpE and dnaJ genes showed high identity with those of other bacterial strains, particularly with those of Bacillus stearothermophilus NUB36, despite an extremely low homology for the corresponding total genomic DNA. There were significant differences in the proline content of the DnaK operon proteins which is closely correlated with the thermostability of enzyme proteins. The proline content was higher in the GrpE, DnaK and DnaJ proteins of the thermophilic as opposed to the mesophilic strains. The overexpression of the B. thermoglucosidasius DnaK protein in Escherichia coli MV1184 results in extreme filamentation without inhibition on cell growth. The B. thermoglucosidasius DnaK protein seemed to exclusively disturb septation in E. coli cells which suggests that it interacts with key protein(s) involved in cell septation.     

Structural Basis of Substrate Selectivity of E. coli Prolidase

Prolidases, metalloproteases that catalyze the cleavage of Xaa-Pro dipeptides, are conserved enzymes found in prokaryotes and eukaryotes. In humans, prolidase is crucial for the recycling of collagen. To further characterize the essential elements of this enzyme, we utilized the Escherichia coli prolidase, PepQ, which shares striking similarity with eukaryotic prolidases. Through structural and bioinformatic insights, we have extended previous characterizations of the prolidase active site, uncovering a key component for substrate specificity. Here we report the structure of E. coli PepQ, solved at 2.0 Å resolution. The structure shows an antiparallel, dimeric protein, with each subunit containing N-terminal and C-terminal domains. The C-terminal domain is formed by the pita-bread fold typical for this family of metalloproteases, with two Mg(II) ions coordinated by five amino-acid ligands. Comparison of the E. coli PepQ structure and sequence with homologous structures and sequences from a diversity of organisms reveals distinctions between prolidases from Gram-positive eubacteria and archaea, and those from Gram-negative eubacteria, including the presence of loop regions in the E. coli protein that are conserved in eukaryotes. One such loop contains a completely conserved arginine near the catalytic site. This conserved arginine is predicted by docking simulations to interact with the C-terminus of the substrate dipeptide. Kinetic analysis using both a charge-neutralized substrate and a charge-reversed variant of PepQ support this conclusion, and allow for the designation of a new role for this key region of the enzyme active site.     

Overproduction and single-step purification of Bacillus stearothermophilus peroxidase in Escherichia coli

Summary The cloned peroxidase gene from Bacillus stearothermophilus was highly expressed in Escherichia coli. Using the high copy number plasmid which is temperature-sensitive and its own strong promoter, this thermostable peroxidase was produced at 28% of the total cell proteins when the cells were grown at 42°C. The enzyme could be easily purified from E. coli by heat treatment and single-column Sephadex G-200 chromatography. From a 200 ml culture, 30 mg of purified enzyme was obtained. The peroxidase produced by E. coli showed a thermostability, haem type and content identical with those of the peroxidase produced by B. stearothermophilus.Offprint requests to: H. Okada     

Biosynthesis of bacterial glycogen: Kinetic studies of a glucose-1-P adenylyltransferase (EC 2.7.7.27) from a glycogen-excess mutant of Escherichia coli B

An Escherichia coli B mutant, CL1136 accumulates glycogen at 3.4 to 4 times the rate observed for the parent E. coli B strain. The glycogen accumulated in the mutant is similar to the glycogen isolated from the parent strain with respect to α- and β-amylolysis, chain length determination and I₂-complex absorption spectra. The CL1136 mutant contains normal glycogen synthase and branching enzyme activity but has an ADPglucose pyrophosphorylase with altered kinetic and allosteric properties. The mutant enzyme has been partially purified and in contrast to the present strain enzyme studied previously, is highly active in the absence of the allosteric activator. The response of the CL1136 enzyme to energy charge has been determined and this enzyme shows appreciable activity at low energy charge values where the E. coli B enzyme is inactive. The response to energy charge for the CL1136 and E. coli B enzymes are correlated with the rates of glycogen accumulation observed in the microorganisms. The regulation of glycogen synthesis in E. coli is to a great extent at the level of ADPglucose pyrophosphorylase; varying concentrations of fructose-P₂ and energy charge determine the rate of ADPglucose and glycogen synthesis. Both the allosteric regulation of ADPglucose pyrophosphorylase as well as the genetic regulations of the synthesis of glycogen biosynthetic enzymes (glycogen synthase and ADPglucose pyrophosphorylase) are involved in the regulation of glycogen accumulation in E. coli B.     

Characterization of an iron sensitive Mud1 mutant in E. coli lacking the ribonucleotide reductase subunit B2

The mutant, generated by a Mud1 insertion, formed long non-viable filaments in the presence of iron and air. Under anaerobic conditions normal growth in the presence of iron was observed. The mutation was mapped by P1 transductions at 48 min on the genetic map of Escherichia coli. By Southern blotting the insertion point was determined to be in nrdB, the structural gene for the ribonucleotide reductase subunit B2. The mutation could be complemented by the cloned nrdB gene. Up to now it was assumed that E. coli possesses only one enzyme for the synthesis of deoxyribunucleotides and only conditional lethal (temperature sensitive) mutants were isolated in nrdB. The insertion of Mud1 in nrdB should lead to a complete loss of the essential B2 subunit. Since the strain was able to grow under anaerobic conditions on minimal medium lacking deoxyribonucleotides an additional pathway for the synthesis of deoxyribonucleotides is postulated.     

Pyrimidine,purine and nitrogen control of cytosine deaminase synthesis in Escherichia coli K12. Involvement of the glnLG and purR genes in the regulation of codA expression

Cytosine deaminase, encoded by the codA gene in Escherichia coli catalyzes the deamination of cytosine to uracil and ammonia. Regulation of codA expression was studied by determining the level of cytosine deaminase in E. coli K12 grown in various defined media. Addition of either pyrimidine or purine nucleobases to the growth medium caused repressed enzyme levels, whereas growth on a poor nitrogen source such as proline resulted in derepression of cytosine deaminase synthesis. Derepression of codA expression was induced by starvation for either uracil or cytosine nucleotides. Nitrogen control was found to be mediated by the glnLG gene products, and purine repression required a functional purR gene product. Studies with strains harbouring multiple mutations affecting both pyrimidine, purine and nitrogen control revealed that the overall regulation of cytosine deaminase synthesis by the different metabolites is cumulative.This paper is dedicated to Professor John Ingraham, Department of Bacteriology, University of California, Davis, on the occasion of his retirement, in recognition of his many contributions in the field of bacterial growth and metabolism     

Secretion of the overproduced periplasmic PhoA protein into the medium and accumulation of its precursor in phoA-transformed Escherichia coli strains: involvement of outer membrane vesicles

Various Escherichia coli strains were transformed by multicopy plasmids pHI-1, pHI-7 and pPHOI carrying the entire regulatory and structural phoA sequences. All transformants with the intact pho regulatory system displayed PhoA oversynthesis and secretion into the medium. They also accumulated the alkaline phosphatase precursor localized in the outer membrane fraction. The dynamics of enzyme synthesis and secretion as well as cell cytomorphology during secretion were studied in strain E. coli K12 802 carrying pHI-7 plasmid. PhoA protein was shown to be selectively released into the medium in vesicles budding from outer membrane.The authors are with the institute of Biochemistry and Physiology of Microorganisms, USSR Academy of Sciences, 142292 Puschino, Moscow Region, USSR.     

L-asparaginase synthesis by Escherichia coli B

We have studied the influence of strain of organism, temperature, and medium on the production of the antileukemic intracellular enzyme L-asparaginase by E. coli B grown in shaken flasks. Five strains of E. coli B exhibited wide differences in their capacities to synthesize the EC-2 form of L-asparaginase active against leukemia. For the most productive strain, when grown in a casein hydrolysate medium, maximal production of L-asparaginase occurred at 25°C. At this temperature, the organism required glycerol, glucose, or other mono-saccharides to synthesize L-asparaginase. Synthesis was stimulated when glycerol was used in place of glucose, but not in its presence. The effect of glycerol on L-asparaginase synthesis was most evident when the cells were grown at 37°C, rather than at 25°C. With 0.25% glucose, cells had a specific activity of 409 I.U./g; with glycerol cells had a specific activity of 553 I.U./g. At 25°C, both cell and L-asparaginase synthesis were increased by the use of 0.25% glycerol resulting in only a slight increase in specific activity of the cells. The addition of zinc, copper, manganese, iron, L-asparagine, L-glutamine, or L-aspartic acid had no effect on L-asparaginase synthesis in the casein hydrolysate medium. L-aspartic acid (10^?2 M) enhanced L-asparaginase synthesis in a synthetic medium that lacked these metals or L-asparagine, L-glutamine, or L-aspartic acid; cells grown under these conditions had a specific activity of 90 I.U./g. In the casein hydrolysate medium, cell morphology was correlated with temperature of incubation.     

The host specificity system inEscherichia coli SK

E. coli SK has its own enzyme system providing DNA host specificity which differs from the known types of specificity inE. coli K12 andE. coli B. Modification and restriction are observed when the PBVI or PBV3 phages are transferred fromE. coli SK toE. coli B or K12 (and back).A methylase has been isolated fromE. coli SK cells and partly purified. This methylase catalyzesin vitro transfer of the labelled methyl groups from S-adenosylmethionine (SAM) to DNA of both phage and tissue origin which gives rise to 5-methylcytosine (5MC) and 6-methylaminopurine (6MAP). The methylase preparations isolated from the cells at the stationary growth have proved to be 1.5–1.7 times as active as the enzyme from the cells at the logarithmic growth stage. The extract ofE. coli SK cells infected with the phage S_D cannot methylate DNAin vitro. This fact is due tode novo synthesis of the enzyme which disintegrates SAM down to 5-methylthioadenosine (5MTA) and homoserine (HS). This enzyme is not found in the cells infected with the S_D phage in the presence of chloroamphenicole. The activity of the enzyme which disintegrates SAM is the highest between the 4th and the 5th minutes of infection. Thus it may be assumed that this enzyme, most probably, is an early virus specific protein and preventsin vivo methylation of the phage DNA.