scholarly journals Isolation of the membrane-bound 26 000-Mr penicillin-binding protein of Streptomyces strain K15 in the form of a penicillin-sensitive d-alanyl-d-alanine-cleaving transpeptidase

1982 ◽  
Vol 207 (1) ◽  
pp. 109-115 ◽  
Author(s):  
M Nguyen-Distèche ◽  
M Leyh-Bouille ◽  
J M Ghuysen
Keyword(s):  
Affinity Chromatography ◽  
Binding Protein ◽  
Peptidase Activity ◽  
Penicillin Binding Protein ◽  
Streptomyces Strain ◽  
Step Procedure ◽  
Membrane Bound ◽  
Sepharose 4B

The membrane-bound, 26 000-Mr penicillin-binding protein of Streptomyces K15 has been isolated in the form of an effective, penicillin-sensitive D-alanyl-D-alanine-cleaving peptidase exhibiting high transpeptidase activity (greater than 95%) and very low carboxy-peptidase activity (less than 5%). The penicillin-binding protein/transpeptidase can be extracted directly from the mycelium with N-cetyl-NNN-trimethylammonium bromide (Cetavlon) and subsequently obtained at 90% purity and with an 8000-fold specific enrichment (when compared with the activity of the isolated membranes) by a two-step procedure involving Sephadex filtration and affinity chromatography on ampicillin-linked CH Sepharose 4B in the presence of detergent. At saturating concentrations of the co-substrates diacetyl-L-Lys-D-Ala-D-Ala and Gly-Gly, the catalytic-centre activity is about 0.3 s-1.

scholarly journals Affinity chromatography and inhibition of chorismate mutase-prephenate dehydrogenase by derivatives of phenylalanine and tyrosine

1977 ◽  
Vol 165 (1) ◽  
pp. 121-126 ◽  
Author(s):  
G D Smith ◽  
D V Roberts ◽  
A Daday
Keyword(s):  
Affinity Chromatography ◽  
Competitive Inhibitor ◽  
Chorismate Mutase ◽  
Prephenate Dehydrogenase ◽  
Step Procedure ◽  
One Step ◽  
High Degree ◽  
Sepharose 4B ◽  
Derivatives Of

Several derivatives of phenylalanine and tyrosine were prepared and tested for inhibition of chorismate mutase-prephenate dehydrogenase (EC 1.3.1.12) from Escherichia coli K12 (strain JP 232). The best inhibitors were N-toluene-p-sulphonyl-L-phenylalanine, N-benzenesulphonyl-L-phenylalanine and N-benzloxycarbonyl-L-phenylalanine. Consequently two compounds, N-toluene-sulphonyl-L-p-aminophenylalanine and N-p-aminobenzenesulphonyl-L-phenylalanine, were synthesized for coupling to CNBr-activated Sepharose-4B. The N-toluene-p-sulphonyl-L-p-aminophenylalanine-Sepharose-4B conjugate was shown to bind the enzyme very strongly at pH 7.5. The enzyme was not eluted by various eluents, including 1 M-NaCl, but could be quantitatively recovered by washing with buffer of pH9. Elution was more effective in the presence of 10 mM-1-adamantaneacetic acid, a competitive inhibitor of the enzyme. This affinity-chromatography procedure results in a high degree of purification of the enzyme and can be used to prepare the enzyme in a one-step procedure from the bacterial crude extract. Such a procedure may therefore prove useful in studying this enzyme in a state that closely resembles that in vivo.

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

1992 ◽  
Vol 282 (3) ◽  
pp. 781-788 ◽  
Author(s):  
B Granier ◽  
C Duez ◽  
S Lepage ◽  
S Englebert ◽  
J Dusart ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Active Site ◽  
Binding Capacity ◽  
Binding Protein ◽  
Secondary Structures ◽  
Streptomyces Lividans ◽  
Dimensional Structure ◽  
Peptidase Activity ◽  
Penicillin Binding Protein

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.

scholarly journals The Enterococcus hirae R40 penicillin-binding protein 5 and the methicillin-resistant Staphylococcus aureus penicillin-binding protein 2′ are similar

1991 ◽  
Vol 280 (2) ◽  
pp. 463-469 ◽  
Author(s):  
A el Kharroubi ◽  
P Jacques ◽  
G Piras ◽  
J Van Beeumen ◽  
J Coyette ◽  
...  
Keyword(s):  
Amino Acid ◽  
Binding Protein ◽  
Water Soluble ◽  
Penicillin Binding Protein ◽  
Enterococcus Hirae ◽  
Methicillin Resistant ◽  
Class B ◽  
Membrane Bound ◽  
Polymerase Chain ◽  
Terminal Domains

The penicillin-resistant Enterococcus hirae R40 has a typical profile of membrane-bound penicillin-binding proteins (PBPs) except that the 71 kDa PBP5 of low penicillin affinity represents about 50% of all the PBPs present. Water-soluble tryptic-digest peptides were selectively produced from PBP5, their N-terminal regions were sequenced and synthetic oligonucleotides were used as primers to generate a 476 bp DNA fragment by polymerase chain reaction. On the basis of these data, the PBP5-encoding gene was cloned in Escherichia coli by using pBR322 as vector. The gene, included in a 7.1 kb insert, had the information for a 678-amino acid-residue protein. PBP5 shows similarity, in the primary structure, with the high-molecular-mass PBPs of class B. In particular, amino acid alignment of the enterococcal PBP5 and the methicillin-resistant staphylococcal PBP2′ generates scores that are 30, for the N-terminal domains, and 53, for the C-terminal domains, standard deviations above that expected for a run of 20 randomized pairs of proteins having the same amino acid compositions as the two proteins under consideration.

scholarly journals The Staphylococcus aureus Chaperone PrsA Is a New Auxiliary Factor of Oxacillin Resistance Affecting Penicillin-Binding Protein 2A

2015 ◽  
Vol 60 (3) ◽  
pp. 1656-1666 ◽  
Author(s):  
Ambre Jousselin ◽  
Caroline Manzano ◽  
Alexandra Biette ◽  
Patricia Reed ◽  
Mariana G. Pinho ◽  
...  
Keyword(s):  
Binding Protein ◽  
Mrna Levels ◽  
Penicillin Binding Protein ◽  
Content Type ◽  
Oxacillin Resistance ◽  
Protein Membrane ◽  
Membrane Bound ◽  
Additional Level ◽  
Auxiliary Factor

Expression of the methicillin-resistantS. aureus(MRSA) phenotype results from the expression of the extra penicillin-binding protein 2A (PBP2A), which is encoded bymecAand acquired horizontally on part of the SCCmeccassette. PBP2A can catalyzedd-transpeptidation of peptidoglycan (PG) because of its low affinity for β-lactam antibiotics and can functionally cooperate with the PBP2 transglycosylase in the biosynthesis of PG. Here, we focus upon the role of the membrane-bound PrsA foldase protein as a regulator of β-lactam resistance expression. Deletion ofprsAaltered oxacillin resistance in three different SCCmecbackgrounds and, more importantly, caused a decrease in PBP2A membrane amounts without affectingmecAmRNA levels. The N- and C-terminal domains of PrsA were found to be critical features for PBP2A protein membrane levels and oxacillin resistance. We propose that PrsA has a role in posttranscriptional maturation of PBP2A, possibly in the export and/or folding of newly synthesized PBP2A. This additional level of control in the expression of themecA-dependent MRSA phenotype constitutes an opportunity to expand the strategies to design anti-infective agents.

scholarly journals Active-site and membrane topology of the DD-peptidase/penicillin-binding protein no. 6 of Enterococcus hirae (Streptococcus faecium) A.T.C.C. 9790

1989 ◽  
Vol 262 (2) ◽  
pp. 457-462 ◽  
Author(s):  
A el Kharroubi ◽  
G Piras ◽  
P Jacques ◽  
I Szabo ◽  
J Van Beeumen ◽  
...  
Keyword(s):  
Active Site ◽  
Binding Capacity ◽  
Binding Protein ◽  
Membrane Topology ◽  
Penicillin Binding Protein ◽  
Enterococcus Hirae ◽  
Serine Residue ◽  
Membrane Bound ◽  
High Degree ◽  
Terminal Appendage

The membrane-bound 43,000-Mr penicillin-binding protein no. 6 (PBP6) of Enterococcus hirae consists of a 30,000-Mr DD-peptidase/penicillin-binding domain and a approximately 130-residue C-terminal appendage. Removal of this appendage by trypsin proteolysis has no marked effect on the catalytic activity and penicillin-binding capacity of the PBP. Anchorage of the PBP in the membrane appears to be mediated by a short 15-20-residue stretch at the C-terminal end of the appendage. The sequence of the 50-residue N-terminal region of the PBP shows high degree of homology with the sequences of the corresponding regions of the PBPs5 of Escherichia coli and Bacillus subtilis. On this basis the active-site serine residue occurs at position 35 in the enterococcal PBP.

scholarly journals General ligands in affinity chromatography. Cofactor–substrate elution of enzymes bound to the immobilized nucleotides adenosine 5′-monophosphate and nicotinamide–adenine dinucleotide

1972 ◽  
Vol 127 (4) ◽  
pp. 625-631 ◽  
Author(s):  
K. Mosbach ◽  
H. Guilford ◽  
R. Ohlsson ◽  
M. Scott
Keyword(s):  
Lactate Dehydrogenase ◽  
Affinity Chromatography ◽  
Cyanogen Bromide ◽  
Competitive Inhibitor ◽  
Single Step ◽  
Lactate Dehydrogenase Activity ◽  
Step Procedure ◽  
The Matrix ◽  
Subsequent Elution ◽  
Sepharose 4B

1. Two different gels have been prepared suitable for the separation of a number of enzymes, in particular NAD+-dependent dehydrogenases, by affinity chromatography. For both the matrix used was Sepharose 4B. For preparation (a), NAD+–Sepharose, 6-aminohexanoic acid has been coupled to the gel by the cyanogen bromide method and then NAD+ was attached by using dicyclohexylcarbodi-imide; for preparation (b), AMP–Sepharose, N6-(6-aminohexyl)-AMP has been coupled directly to cyanogen bromide-activated gel. 2. Affinity columns of both gels retain only the two enzymes when a mixture of bovine serum albumin, lactate dehydrogenase and glyceraldehyde 3-phosphate dehydrogenase is applied. Subsequent elution with the cofactor NAD+ yields glyceraldehyde 3-phosphate dehydrogenase whereas lactate dehydrogenase is eluted by applying the same molarity of the reduced cofactor. 3. The binding of both glyceraldehyde 3-phosphate dehydrogenase and lactate dehydrogenase to the gel tested, AMP–Sepharose, is strong enough to resist elution by gradients of KCl of up to at least 0.5m. A 0.0–0.15m gradient of the competitive inhibitor salicylate, however, elutes both enzymes efficiently and separately. 4. The elution efficiency of lactate dehydrogenase from AMP–Sepharose has been examined by using a series of eluents under comparable conditions of concentration etc. The approximate relative efficiencies are: 0 (lactate); 0 (lactate+semicarbazide); 0 (0.5mm-NAD+); 80 (lactate+NAD+); 95 (lactate+semicarbazide+NAD+); 100 (0.5mm-NADH). 5. All contaminating lactate dehydrogenase activity can be removed from commercially available crude pyruvate kinase in a single-step procedure by using AMP–Sepharose.

scholarly journals Secretion by overexpression and purification of the water-soluble Streptomyces K15 dd-transpeptidase/penicillin-binding protein

1992 ◽  
Vol 288 (1) ◽  
pp. 87-91 ◽  
Author(s):  
P Palomeque-Messia ◽  
V Quittre ◽  
M Leyh-Bouille ◽  
M Nguyen-Distèche ◽  
C J L Gershater ◽  
...  
Keyword(s):  
Culture Supernatant ◽  
Binding Capacity ◽  
Binding Protein ◽  
Water Soluble ◽  
Appreciable Amount ◽  
Soluble Enzyme ◽  
Penicillin Binding Protein ◽  
Wild Type ◽  
Wild Type Enzyme ◽  
Membrane Bound

Though synthesized with a cleavable signal peptide and devoid of membrane anchors, the 262-amino-acid-residue Streptomyces K15 DD-transpeptidase/penicillin-binding protein is membrane-bound. Overexpression in Streptomyces lividans resulted in the export of an appreciable amount of the synthesized protein (4 mg/litre of culture supernatant). The water-soluble enzyme was purified close to protein homogeneity with a yield of 75%. It requires the presence of 0.5 M-NaCl to remain soluble. It is indistinguishable from the detergent-extract wild-type enzyme with respect to molecular mass, thermostability, transpeptidase activity and penicillin-binding capacity.

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