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PubMed2005

Identification and molecular characterization of an N-acetylmuramyl-L-alanine amidase Sle1 involved in cell separation of Staphylococcus aureus.

We purified a peptidoglycan hydrolase involved in cell separation from a Staphylococcus aureus atl null mutant and identified its gene. Characterization of the gene product shows a 32 kDa N-acetylmuramyl-L-alanine amidase that we designated Sle1. Analysis of peptidoglycan digests showed Sle1 preferentially cleaved N-acetylmuramyl-L-Ala bonds in dimeric cross-bridges that interlink the two murein strands in the peptidoglycan. An insertion mutation of sle1 impaired cell separation and induced S. aureus to form clusters suggesting Sle1 is involved in cell separation of S. aureus. The Sle1 mutant revealed a significant decrease in pathogenesis using an acute infection mouse model. Atl is the major autolysin of S. aureus, which has been implicated in cell separation of S. aureus. Generation of an atl/sle1 double mutant revealed that the mutant cell separation was heavily impaired suggesting that S. aureus uses two peptidoglycan hydrolases, Atl and Sle1, for cell separation. Unlike Atl, Sle1 is not directly involved in autolysis of S. aureus.

Amino Acid Motifs↗

PubMed1989

Analysis of N-acetyl-4-O-acetylneuraminic-acid-containing N-linked carbohydrate chains released by peptide-N4-(N-acetyl-beta-glucosaminyl)asparagine amidase F. Application to the structure determination of the carbohydrate chains of equine fibrinogen.

The carbohydrate chains of equine fibrinogen were enzymatically released by peptide-N4-(N-acetyl-beta-glucosaminyl)asparagine amidase F. The oligosaccharides obtained were fractionated by a combination of FPLC and HPLC and analyzed by 500-MHz 1H-NMR spectroscopy. Four monosialo and four disialo diantennary N-acetyllactosamine type of carbohydrate chains occur: (formula; see text)

Acetylation↗

PubMed1991

Peptide-N4-(N-acetyl-beta-glucosaminyl)asparagine amidase F cannot release glycans with fucose attached alpha 1----3 to the asparagine-linked N-acetylglucosamine residue.

The ability of peptide-N4-(N-acetyl-beta-glucosaminyl)asparagine amidase F (PNGase F) from Flavobacterium meningosepticum and PNGase A from sweet almonds to deglycosylate N-glycopeptides and N-glycoproteins from plants was compared. Bromelain glycopeptide and horseradish peroxidase-C glycoprotein, which contain xylose linked beta 1----2 to beta-mannose and fucose linked alpha 1----3 to the innermost N-acetylglucosamine, were used as substrates. In contrast to PNGase A, the enzyme from F. meningosepticum did not act upon these substrates even at concentrations 100-fold higher than required for complete deglycosylation of commonly used standard substrates. After removal of alpha 1----3-linked fucose from the plant glycopeptide and glycoprotein by mild acid hydrolysis, they were readily degraded by PNGase F at moderate enzyme concentrations. Hence we conclude that alpha 1----3 fucosylation of the inner N-acetylglucosamine impedes the enzymatic action of PNGase F. Knowledge of this limitation of the deglycosylation potential of PNGase F may turn it from a pitfall into a useful experimental tool.

Acetylglucosamine↗

PubMed2002

Production of glycosylated thermostable Providencia rettgeri penicillin G amidase in Pichia pastoris.

Penicillin G amidase from Providencia rettgeri is a heterodimer of 92 kDa. We have previously expressed the Pr. rettgeri pac gene coding for this enzyme in Saccharomyces cerevisiae, and now we report the expression and characterization in the methylotrophic yeast Pichia pastoris. The recombinant catalytically active enzyme (rPAC(Pr)) was secreted from shake flask-grown P. pastoris cells into the medium at a level of approximately 0.18 U ml(-1). This yield of rPAC(Pr) was higher, by two orders of magnitude, than that obtained using a single-copy expression plasmid in S. cerevisiae. In addition, the secreted recombinant enzyme was entirely N-glycosylated. The recombinant PAC(Pr) was further characterized in terms of specific activity, kinetic parameters and thermostability. Except the significantly higher thermostability of the glycosylated rPAC(Pr) produced in P. pastoris, the other parameters were very similar to those of the corresponding non-glycosylated enzymes produced in bacteria or in S. cerevisiae. The higher thermostability of this recombinant enzyme has a clear industrial advantage.

Enzyme Stability↗

PubMed1998

The two-component lysis system of Staphylococcus aureus bacteriophage Twort: a large TTG-start holin and an associated amidase endolysin.

The lysis genes of the virulent Staphylococcus aureus bacteriophage Twort were cloned and their nucleotide sequences determined. The endolysin gene plyTW encodes a 53.3-kDa protein, whose catalytic site is located in the amino-terminal domain. An enzymatically active fragment (N-terminal 271 amino acids) was overexpressed in Escherichia coli and partially purified. The enzyme rapidly cleaves staphylococcal peptidoglycan, and was shown to act as N-acetylmuramoyl-L-alanine amidase (EC 3.5.1.28). Significant sequence homology to the specific cell wall targeting domain of lysostaphin was observed in a 101-amino acid C-terminal overlap. However, we found that the large C-terminal portion (63%, 295 aa) of PlyTW is not required for staphylolytic activity. Located upstream of and overlapping plyTW by 35 bp in a different reading frame (+1), we identified holTW, which starts with a single TTG triplet. The gene specifies a 185-amino acid (20.5 kDa) holin protein, which features two potential hydrophobic, antiparallel transmembrane domains, and a highly charged, acidic C-terminus. HolTW is the largest class II holin described to date. It can substitute for the defective allele in phase lambda S' amber mutants, both in trans from an expression plasmid, and from within gt11::holTW. The proposed function is the formation of unspecific membrane lesions to promote access of the endolysin to the bacterial peptidoglycan.

Amino Acid Sequence↗

PubMed2000

The staphylokinase gene is located in the structural gene encoding N-acetylmuramyl-L-alanine amidase in methicillin-resistant Staphylococcus aureus.

The nucleotide sequence of a 15600-bp DNA fragment containing the staphylokinase gene (sakNU3-1) of methicillin-resistant Staphylococcus aureus (MRSA) NU3-1 was determined. The sak gene was found within the ply gene encoding N-acetylmuramyl-L-alanine amidase and thus the ply gene should be inactivated. In the flanking region of the sak gene, the tandem repeat sequences (GAAGTGTT and GAATGGTT) were present as possible junction points between the sak and ply genes. No sequences characteristic of the presence of an IS-like element were found. Upstream from the ply gene, the kdpA, kdpB and kdpC homologues were present. Downstream from the ply gene, the tagA, tagH and tagG homologues were present. The sak gene was inserted into the same position of ply in 5/6 of sak(+) MRSA isolates with different genotypes. In all of these sak(+) isolates, Sak was detected in the culture supernatant.

Cloning, Molecular↗

PubMed1985

Molecular cloning of Bacillus sphaericus penicillin V amidase gene and its expression in Escherichia coli and Bacillus subtilis.

The Bacillus sphaericus gene coding for penicillin V amidase, which catalyzes the hydrolysis of penicillin V to yield 6-aminopenicillanic acid and phenoxyacetic acid, has been isolated by molecular cloning in Escherichia coli. The gene is contained within a 2.2-kilobase HindIII-PstI fragment and is expressed when transferred into E. coli and Bacillus subtilis. The expression in B. subtilis carrying the recombinant plasmid is approximately two times higher than in the original B. sphaericus strain. A comparison of the purified enzyme from B. sphaericus and the expressed gene product in E. coli minicells suggests that the native enzyme consists of four identical subunits, each with a molecular weight of 35,000.

Amidohydrolases↗

PubMed2003

Improvement of posttranslational bottlenecks in the production of penicillin amidase in recombinant Escherichia coli strains.

Using periplasmic penicillin amidase (PA) from Escherichia coli ATCC 11105 as a model recombinant protein, we reviewed the posttranslational bottlenecks in its overexpression and undertook attempts to enhance its production in different recombinant E. coli expression hosts. Intracellular proteolytic degradation of the newly synthesized PA precursor and translocation through the plasma membrane were determined to be the main posttranslational processes limiting enzyme production. Rate constants for both intracellular proteolytic breakdown (k(d)) and transport (k(t)) were used as quantitative tools for selection of the appropriate host system and cultivation medium. The production of mature active PA was increased up to 10-fold when the protease-deficient strain E. coli BL21(DE3) was cultivated in medium without a proteinaceous substrate, as confirmed by a decrease in the sum of the constants k(d) and k(t). The original signal sequence of pre-pro-PA was exchanged with the OmpT signal peptide sequence in order to increase translocation efficiency; the effects of this change varied in the different E. coli host strains. Furthermore, we established that simultaneous coexpression of the OmpT pac gene with some proteins of the Sec export machinery of the cell resulted in up to threefold-enhanced PA production. In parallel, we made efforts to increase PA flux via coexpression with the kil gene (killing protein). The primary effects of the kil gene were the release of PA into the extracellular medium and an approximately threefold increase in the total amount of PA produced per liter of bacterial culture.

Bacterial Proteins↗

PubMed1983

Mutation of the N-acetylmuramyl-L-alanine amidase gene of Escherichia coli K-12.

Mutants of Escherichia coli with very low N-acetylmuramyl-L-alanine amidase activity were isolated. The gene amiA responsible for most of this enzyme activity was mapped at 51 min on the E. coli chromosome, with the most plausible gene order assumed to be amiA pts(H or I) purC. The mutant phenotype was recessive and physiologically undiscernible.

Amidohydrolases↗

PubMed1999

Folate-targeted enzyme prodrug cancer therapy utilizing penicillin-V amidase and a doxorubicin prodrug.

In antibody-targeted enzyme prodrug therapy, a monoclonal antibody (mAb) covalently linked to an enzyme is commonly exploited to concentrate the enzyme on the tumor cell surface prior to administration of a relatively nontoxic prodrug. The tumor-localized enzyme then converts the prodrug into a cytotoxic agent, which in turn diffuses into the tumor causing localized cell death. In this paper, we have substituted folic acid for the mAb as a mean of delivering an attached enzyme, penicillin-V amidase (PVA), to folate receptor (FR)-positive tumor cells. The enzyme PVA is capable of converting a doxorubicin-N-p-hydroxyphenoxyacetamide prodrug (DPO) into its potent parent drug, doxorubicin. For PVA targeting, each PVA molecule was covalently labeled with three molecules of folic acid via the formation of amide bonds. In vitro binding assays showed that folate-PVA-125I conjugates bind specifically to KB cells (FR-positive tumor cells) but not to A549 cells (FR-negative tumor cells). Moreover, in a series of in vitro cytotoxicity tests, folate-PVA conjugates were found to kill folate receptor positive but not receptor negative cells, and when bound to FR-positive cells, folate-PVA conjugates rendered the DPO prodrug as toxic as free doxorubicin (IC50, approximately 0.6 microM). Finally, preliminary in vivo plasma clearance studies in normal mice revealed that i.v. administered folate-PVA-125I and PVA-125I are both cleared from the blood within a 24 h time period, removing concern that nonspecifically trapped folate-PVA might activate prodrug in nontargeted tissues. In view of the fact that only a small number of folate-PVA molecules are required to mediate killing of target cells in vitro, these data argue that folate-targeted enzyme prodrug therapy should be considered for tumor eradication in vivo.

Animals↗

PubMed1991

Structure determination by 1H NMR spectroscopy of (sulfated) sialylated N-linked carbohydrate chains released from porcine thyroglobulin by peptide-N4-(N-acetyl-beta-glucosaminyl)asparagine amidase-F.

The N-linked carbohydrate chains of porcine thyroglobulin were released by peptide-N4-(N-acetyl-beta-glucosaminyl)asparagine amidase-F (PNGase-F). The resulting oligosaccharides were fractionated by a combination of fast protein liquid chromatography and high performance liquid chromatography and analyzed by 500-MHz 1H NMR spectroscopy. The major acidic compounds are mono- and disialylated, fucosylated diantennary compounds terminated with alpha(2-6)-linked sialic acid on the Man alpha(1-3) branch. The Man alpha(1-6) branch shows a large heterogeneity. It can be terminated with Man-4', GlcNAc-5', or Gal-6', whereas the Gal-6' residue may be extended with Gal alpha(1-3), NeuAc alpha(2-3), or Sia alpha (2-6). In the major structures 8% of alpha(2-6)-linked sialic acid was found as NeuGc instead of NeuAc. The main compounds have sulfated homologues bearing a sulfate group (6-20%) at C-3 of Gal-6' or at C-6 of GlcNAc-5 as follows. [formula: see text]

Amidohydrolases↗

PubMed1990

Cloning and expression of peptide-N4-(N-acetyl-beta-D-glucosaminyl)asparagine amidase F in Escherichia coli.

The peptide-N4-(N-acetyl-beta-D-glucosaminyl) asparagine amidase F (PNGase F) gene from Flavobacterium meningosepticum was cloned into a high copy number Escherichia coli plasmid. Levels of PNGase F activity produced in cultures of the recombinant strain were up to 100-fold higher than those obtained in cultures of F. meningosepticum. The complete PNGase F gene sequence was determined. Comparison of the predicted amino acid sequence of pre-PNGase F to the N-terminal sequence of the native mature enzyme indicates that the protein is synthesized with a 40-amino acid signal sequence that is removed during secretion in F. meningosepticum. The recombinant PNGase F produced in E. coli is a mixture of products comprised predominantly of two proteins with molecular masses of 36.3 and 36.6 kDa. These proteins have a higher apparent molecular mass than the 34.7-kDa native enzyme. N-terminal amino acid sequencing demonstrated that these higher molecular mass products result from cleavage of the pre-PNGase F in E. coli upstream of the native N terminus. The PNGase F gene was engineered to encode a preenzyme that was processed in E. coli to give an N terminus identical to that of the native enzyme. Purified preparations of this form of recombinant PNGase F were shown to be suitable for glycoprotein analyses since they possess no detectable endo-beta-N-acetylglucosaminidase F, exoglycosidase, or protease activity.

Amidohydrolases↗

PubMed1978

[Study of E. coli penicillin amidase. The pH dependence of the equilibrium constant of ampicillin enzymatic hydrolysis].

The equilibrium parameters of the hydrolysis of ampicillin catalysed by penicillin amidase were determined within the pH range of 4.5 to 5.5. The values of the ionization constants of the carboxy group of D-(-)-ALPHA-AMINOPHENYLACETIC ACID (PK1=1.80) and amino group of 6-aminopenicillanic acid (pK2=4.60) were estimated and pH-dependence of the effective free energy of ampicillin hydrolysis was calculated. It was shown that the thermodynamic optimum of ampicillin synthesis was at 3.20 (the value of the effective free energy under the experimental conditions was 3.27 kcal/mole). The value of the "true", pH-independent free energy of hydrolysis (deltasigma) of the amide bond in the ampicillin molecule was determined to be equal to 9.72 kcal/mole. The thermodynamic parameters of ampicillin and benzylpenicillin hydrolysis were compared. The amino group in the alpha-position of phenylacetic acid was shown to have a significant effect on the values of "true" free energy of hydrolysis of the penicillin amide bond and free ionization energy in the system.

Amidohydrolases↗

PubMed1985

[Phenotypical curing of Streptococcus pneumoniae treated with amidase induced by the Dp-1 bacteriophage].

A phage-associated murein hydrolase activity (PAL) induced in an autolysis-defective mutant of Streptococcus pneumoniae infected with the bacteriophage Dp-1 has been recently isolated and purified to electrophoretic homogeneity as well as biochemically characterized as an endo-N-acetyl-muramyl-L-alanine amidase (1, 3, 4). The PAL and the inactive form (E-form) of the host cell autolysin show a remarkable biochemical similarity, although they differ in their immunological characteristics. The PAL was adsorbed onto a live, defective mutant of pneumococcus (cwl) and such cells reverted to the wild type phenotype ("cured" cells) in some important characteristics present in the wild type strain (R6), as: i) lysis of the culture in the stationary phase, ii) protoplast formation by hypertonic sucrose, and iii) bacteriolytic response against the penicillin in contrast with the bacteriostatic response of the "non-cured" cwl. The adsorbed enzyme segregates during growth of the "cured" cells. Our results demonstrate that PAL acts in the phenotypically "cured" cells in a similar way to that previously described for the host enzyme, and also confirm the finding that the autolysins play a direct role in the irreversible effects produced in S. pneumoniae by the betalactamic antibiotics.

Amidohydrolases↗

PubMed1984

[Isolation of a highly pure preparation of cephalexin amidase from bacteria of the genus Xanthomonas].

A procedure for highly purified cephalexin amidase of Xanthomonas was developed. It consists of preparation of a cell-free extract of the culture after cell disintegration, precipitation with ammonium sulfate, dissolution, concentration and elimination of ballast proteins, gel filtration on Sephadex G-25, sorption of ballast proteins on DEAE cellulose and chromatography on KM-cellulose. The enzyme yield is 45-55 per cent. The purity level is 80-90-fold.

Amidohydrolases↗

PubMed1976

[Study of E. coli penicillin amidase. The pH dependence of the equilibrium constant of the enzymatic hydrolysis of benzylpenicillin].

The equilibrium constant for penicillin amidase-catalyzed hydrolysis of benzylpenicillin(Keg =3.00 +/- 0.24 x 10(-3) M at pH 5.0) and the ionization constants for phenylacetic acid (PAA) and the amino groups of 6-aminopenicillanic acid (6-APA) were determined (4.20 and 4.60 under conditions of the kinetic experiments respectively). The experimental data at pH 6.0 satisfactorily correlated with the theoretical pH-dependence for Keg constructed according to the hypothesis that benzylpenicillin synthesis has a thermodynamic optimum at pH 4.4 equal to a half-sum of the pK values for the carboxylic and amino groups of the PAA and 6-APA respectively.

Amidohydrolases↗

PubMed1997

Expression and intracellular localization of the human N-acetylmuramyl-L-alanine amidase, a bacterial cell wall-degrading enzyme.

N-acetylmuramyl-L-alanine amidase (NAMLAA) specifically degrades peptidoglycan, which is a major component of bacterial cell walls with strong inflammatory properties. For instance, peptidoglycan is capable of stimulating peripheral blood cells to release pro-inflammatory cytokines and is capable of inducing chronic arthritis in an animal model. In a previous study we found that degradation of peptidoglycan by purified NAMLAA reduced its inflammatory effects. To determine where NAMLAA is located in tissues, monoclonal antibodies against purified NAMLAA were produced for use in immunohistochemistry, immunoelectron microscopy, flow cytometric analysis, and Western blotting. The immunohistochemical studies showed NAMLAA-positive cells in human spleen, liver, arthritic synovial tissues, and lymph nodes. In flow cytometric studies of blood and bone marrow, neutrophilic and eosinophilic granulocytes proved to be positive. Monocytes were negative, although they do contain lysozyme, the other important peptidoglycan-degrading enzyme. However, mature macrophages obtained by bronchoalveolar lavage and subsequent selection based on autofluorescence did possess NAMLAA. In immunocytochemical staining of blood smears, thrombocytes were also positive for NAMLAA. Western blot analysis and immunoelectron microscopy of neutrophils and eosinophils showed that NAMLAA is located in azurophilic granules of neutrophils and in secretory vesicles and crystalloid-containing granules of eosinophils. Flow cytometric analysis of blood and bone marrow from different French-American-British-classified acute myeloid leukemia (AML) patients showed that AML-M2 myeloblasts were the first in the granulocyte maturation lineage that were positive for NAMLAA. The more immature AML, such as AML-M0 and AML-M1, did not express NAMLAA. CD15- and CD13-negative megakaryoblasts, corresponding to AML-M7, were also positive for NAMLAA. The expression pattern of NAMLAA in the myeloid lineage suggests that the monoclonal antibody AAA4, recognizing NAMLAA, is useful for discrimination between AML in the monocyte lineage and in the granulocyte lineage.

Acute Disease↗

PubMed2003

Direct hydroxide attack is a plausible mechanism for amidase antibody 43C9.

Direct hydroxide attack on the scissile carbonyl of the substrate has been suggested as a likely mechanism for esterase antibodies elicited by phosphonate haptens, which mimic the transition states for the alkaline hydrolysis of esters.1 The unique amidase activity of esterase antibody 43C9 has been attributed to nucleophilic attack by an active-site histidine residue.2 Yet, the active site of 43C9 is strikingly similar to those of other esterase antibodies, particularly 17E8. We have carried out quantum mechanical calculations, molecular dynamics simulations, and free energy calculations to assess the mechanism involving direct hydroxide attack for 43C9. Results support this mechanism and suggest that the mechanism is plausible for other antiphosphonate antibodies that catalyze the hydrolysis of (p-nitro)phenyl esters.

Amidohydrolases↗