scholarly journals Purification of β-lactamases by affinity chromatography on phenylboronic acid-agarose

1984 ◽  
Vol 221 (2) ◽  
pp. 505-512 ◽  
Author(s):  
S J Cartwright ◽  
S G Waley
Keyword(s):  
Affinity Chromatography ◽  
Boronic Acid ◽  
Phenylboronic Acid ◽  
Boronic Acids ◽  
High Yield ◽  
Beta Lactamase ◽  
Beta Lactamases ◽  
Cell Extracts ◽  
Pseudomonas Maltophilia ◽  
One Step

Several beta-lactamases, enzymes that play an important part in antibiotic resistance, have been purified by affinity chromatography on boronic acid gels. The procedure is rapid, appears to be selective for beta-lactamases, and allows a one-step purification of large amounts of enzyme from crude cell extracts. We have found the method useful for any beta-lactamase that is inhibited by boronic acids. Two kinds of boronic acid column have been prepared, the more hydrophobic one being reserved for those beta-lactamases that bind boronic acids relatively weakly. beta-Lactamase I from Bacillus cereus, P99 beta-lactamase and K 1 beta-lactamase from Gram-negative bacteria are among the better-known beta-lactamases that have been purified by this method. The procedure has also been used to purify a novel beta-lactamase from Pseudomonas maltophilia in high yield; the enzyme has an exceptionally broad substrate profile and hydrolyses monocyclic beta-lactams such as azthreonam and desthiobenzylpenicillin.

scholarly journals An overview of the kinetic parameters of class B β-lactamases

1993 ◽  
Vol 291 (1) ◽  
pp. 151-155 ◽  
Author(s):  
A Felici ◽  
G Amicosante ◽  
A Oratore ◽  
R Strom ◽  
P Ledent ◽  
...  
Keyword(s):  
Aeromonas Hydrophila ◽  
Catalytic Properties ◽  
Beta Lactamase ◽  
Beta Lactamases ◽  
Class B ◽  
Pseudomonas Maltophilia ◽  
Purification Methods ◽  
Unique Specificity ◽  
Chromatography Step ◽  
Sequence Similarities

The catalytic properties of three class B beta-lactamases (from Pseudomonas maltophilia, Aeromonas hydrophila and Bacillus cereus) were studied and compared with those of the Bacteroides fragilis enzyme. The A. hydrophila beta-lactamase exhibited a unique specificity profile and could be considered as a rather specific ‘carbapenemase’. No relationships were found between sequence similarities and catalytic properties. The problem of the repartition of class B beta-lactamases into sub-classes is discussed. Improved purification methods were devised for the P. maltophilia and A. hydrophila beta-lactamases including, for the latter enzyme, a very efficient affinity chromatography step on a Zn(2+)-chelate column.

scholarly journals The production and molecular properties of the zinc β-lactamase of Pseudomonas maltophilia IID 1275

1985 ◽  
Vol 229 (3) ◽  
pp. 791-797 ◽  
Author(s):  
R Bicknell ◽  
E L Emanuel ◽  
J Gagnon ◽  
S G Waley
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Thiol Group ◽  
The Other ◽  
Beta Lactamase ◽  
Beta Lactamases ◽  
Zinc Enzyme ◽  
Pseudomonas Maltophilia ◽  
Measurable Rate ◽  
Beta Lactamase Ii

The production and purification of a tetrameric zinc beta-lactamase from Pseudomonas maltophilia IID 1275 were greatly improved. Three charge variants were isolated by chromatofocusing. The subunits each contain two atomic proportions of zinc and (in two of the variants) one residue of cysteine. The thiol group is not required for activity, nor does it appear to bind to the metal. Replacement of zinc by cobalt, cadmium or nickel takes place at a measurable rate, and gives enzymes that are less active than the zinc enzyme. The properties of this enzyme differ from those of the other known zinc beta-lactamase, beta-lactamase II from Bacillus cereus. The amino acid sequence of the N-terminal 32 residues was determined; there is no similarity to the N-terminal sequences of other beta-lactamases.

scholarly journals Catabolism of Arylboronic Acids by Arthrobacter nicotinovorans Strain PBA

2003 ◽  
Vol 69 (7) ◽  
pp. 4263-4267 ◽  
Author(s):  
Ana C. Negrete-Raymond ◽  
Barbara Weder ◽  
Lawrence P. Wackett
Keyword(s):  
Carbon Atom ◽  
Oxygen Atom ◽  
Boronic Acid ◽  
Phenylboronic Acid ◽  
Boronic Acids ◽  
Ring Carbon ◽  
Arylboronic Acids ◽  
Arthrobacter Nicotinovorans ◽  
Ring Carbon Atom

ABSTRACT Arthrobacter sp. strain PBA metabolized phenylboronic acid to phenol. The oxygen atom in phenol was shown to be derived from the atmosphere using 18O2. 1-Naphthalene-, 2-naphthalene-, 3-cyanophenyl-, 2,5-fluorophenyl-, and 3-thiophene-boronic acids were also transformed to monooxygenated products. The oxygen atom in the product was bonded to the ring carbon atom originally bearing the boronic acid substituent with all the substrates tested.

scholarly journals Selective Chemical Modification of DNA with Boronic Acids by On-Column CuAAc Reactions

Synthesis ◽  
2020 ◽  
Author(s):  
Michael Smietana ◽  
Mégane Debiais ◽  
Jean-Jacques Vasseur ◽  
Sabine Müller
Keyword(s):  
Boronic Acid ◽  
Phenylboronic Acid ◽  
Boronic Acids ◽  
Acid Moiety ◽  
Copper Salts ◽  
Rna Sequences ◽  
New Methods ◽  
Dna And Rna ◽  
Selective Incorporation ◽  
Oligonucleotide Conjugates

The use of the Cu(I)-catalyzed azide–alkyne cycloaddition (CuAAC) reaction for the preparation of oligonucleotide conjugates is by now familiar. However, the selective introduction of boronic acids into DNA and RNA sequences by CuAAC reactions has long been considered impossible due to the incompatibility of the boronic acid moiety with copper salts. Here we describe two new methods for the selective on-column functionalization of oligonucleotides with boronic acids via two different CuAAC reactions. The first one allows the introduction of a phenylboronic acid at the 5′-extremity of oligonucleotides, while the selective intrastrand positioning of the modification can be achieved with the second one. Both methods were applied to the DNA and RNA series (up to a 20-mer) with good isolated yields and excellent purities. These results illustrate the potential of the reported methods for selective incorporation of boronic acids into oligonucleotides.

scholarly journals The inhibition of class C β-lactamases by boronic acids

1983 ◽  
Vol 209 (1) ◽  
pp. 229-233 ◽  
Author(s):  
T Beesley ◽  
N Gascoyne ◽  
V Knott-Hunziker ◽  
S Petursson ◽  
S G Waley ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Pseudomonas Aeruginosa ◽  
Hydroxy Group ◽  
Phenylboronic Acid ◽  
Boronic Acids ◽  
The Other ◽  
Serine Residue ◽  
Reversible Inhibitors ◽  
Beta Lactamases ◽  
Class C

Aromatic boronic acids are reversible inhibitors of the recently classified class C beta-lactamases. The boronic acids studied include ortho-, meta- and para-methyl-, -hydroxymethyl- and -formyl-phenylboronic acid. The beta-lactamases were chromosomally-encoded enzymes, one from Pseudomonas aeruginosa, and the other specified by the ampC gene of Escherichia coli. The inhibition may be correlated with our finding that these beta-lactamases are serine enzymes, i.e. their function entails the hydroxy group of a serine residue acting as a nucleophile.

scholarly journals β-lactamase inhibitors. The inhibition of serine β-lactamases by specific boronic acids

1988 ◽  
Vol 251 (2) ◽  
pp. 453-459 ◽  
Author(s):  
I E Crompton ◽  
B K Cuthbert ◽  
G Lowe ◽  
S G Waley
Keyword(s):  
Active Site ◽  
Ph Dependence ◽  
Boronic Acids ◽  
Penicillin G ◽  
Side Chain ◽  
Beta Lactamase ◽  
Beta Lactamases ◽  
Kinetics Of ◽  
Kinetics Of Inhibition ◽  
Step Mechanism

Many beta-lactamases have active-site serine residues, and are competitively inhibited by boronic acids. Hitherto, the boronic acids used have lacked any structural resemblance to the substrates of beta-lactamases. Phenylacetamidomethaneboronic acid, trifluoroacetamidomethaneboronic acid and 2,6-dimethoxybenzamidomethaneboronic acid have now been synthesized. The first of these contains the side-chain moiety of penicillin G, and the last that of methicillin. The pH-dependence of binding of the first inhibitor to beta-lactamase I from Bacillus cereus revealed pK values of 4.7 and 8.2 for (presumably) active-site groups in the enzyme. The kinetics of inhibition were studied by cryoenzymology and by stopped-flow spectrophotometry. These techniques provided evidence for a two-step mechanism of binding of the first two boronic acids mentioned above to beta-lactamase I, and for benzeneboronic acid to a beta-lactamase from Pseudomonas aeruginosa. The slower step is probably associated with a change in enzyme conformation as well as the formation of an O-B bond between the active-site serine hydroxy group and the boronic acid.

scholarly journals Organic Boronate Affinity Sorbent for Capture of cis-Diol Containing Compounds Eman Alzahrani

2019 ◽  
Vol 31 (9) ◽  
pp. 2073-2082
Author(s):  
Eman Alzahrani
Keyword(s):  
Affinity Chromatography ◽  
In Situ Polymerization ◽  
Phenylboronic Acid ◽  
Covalent Bond ◽  
Study Results ◽  
Bet Analysis ◽  
Ethylene Dimethacrylate ◽  
One Step ◽  
Uv Lamp ◽  
Poly Ethylene

Boronate affinity chromatography (BAC) is argued to be a critical tool in specific capture and separation of cis-diol containing compounds. In present study, organic boronate affinity monolith poly(3-acrylamido phenylboronic acid-co-ethylene dimethacrylate) (AAPBA-co-EDMA) is prepared through one-step in situ polymerization procedure within a micropipette through the application of a pre-polymerization mixture which contains functional monomer (3-acrylamido phenylboronic acid), cross-linker (ethylene dimethacrylate), porogenic solvent (methanol with poly ethylene glycol) and initiator (2,2-dimethoxy-2-phenyl-acetophenone). Following the optimization of time exposure to UV lamp with 365 nm, the macroporous organic boronate monolith was selected. Several approaches including SEM and BET analysis, FT-IR spectroscopy and measuring contact angle were applied in the characterization of the morphology of the monolith. Several cis-diol compounds that include catechol and galactose are applied in the assessment of the boronate affinity of the organic monolithic material. Additionally, the capture of glucose from urine sample is also conducted. The basic principle of the approach is that boronic acid forms covalent bond with cis-diols in basic solutions whereas the ester bonds are dissociated under acidic media. By using the study results, monolith demonstrate good selectivity towards cis-diol containing compounds. Due to the hydrophilic property of monolith, the affinity chromatography monolith can be performed for several cis-diol compounds including glycoproteins and nucleosides. Also, fabrication of the organic boronate monolithic in microfluidic equipment is essential in facilitating the extraction of boronate affinity using small-volume samples.

scholarly journals Characterization of peptidyl boronic acid inhibitors of mammalian 20 S and 26 S proteasomes and their inhibition of proteasomes in cultured cells

2000 ◽  
Vol 346 (2) ◽  
pp. 447-454 ◽  
Author(s):  
Robert C. GARDNER ◽  
Stephen J. ASSINDER ◽  
Gary CHRISTIE ◽  
Grant G. F. MASON ◽  
Roger MARKWELL ◽  
...  
Keyword(s):  
Protein Degradation ◽  
Boronic Acid ◽  
Cultured Cells ◽  
Gel Filtration ◽  
Boronic Acids ◽  
Cell Extracts ◽  
Catalytic Sites ◽  
Ic50 Values ◽  
In Cells

Proteasomes are large multisubunit proteinases which have several distinct catalytic sites. In this study a series of di- and tri-peptidyl boronic acids have been tested on the chymotrypsin-like activity of purified mammalian 20 S and 26 S proteasomes assayed with succinyl-Leu-Leu-Val-Tyr-amidomethylcoumarin (suc-Leu-Leu-Val-Tyr-AMC) as substrate. The inhibition of 20 S proteasomes is competitive but only slowly reversible. The Ki values for the best inhibitors were in the range 10-100 nM with suc-Leu-Leu-Val-Tyr-AMC as substrate, but the compounds tested were much less effective on other proteasome activities measured with other substrates. Free boronic acid inhibitors exhibited equivalent potency to their pinacol esters. Both benzoyl (Bz)-Phe-boroLeu and benzyloxycarbonyl (Cbz)-Leu-Leu-boroLeu pinacol ester inhibited 20 S and 26 S proteasomes with non-ideal behaviour, differences in inhibition of the two forms of proteasomes becoming apparent at high inhibitor concentrations (above 3×Ki). Both of these compounds were also potent inhibitors of 20 S and 26 S proteasomes in cultured cells. However, gel filtration of cell extracts prepared from cells treated with radiolabelled phenacetyl-Leu-Leu-boroLeu showed that only 20 S proteasomes were strongly labelled, demonstrating differences in the characteristics of inhibition of 20 S and 26 S proteasomes. The usefulness of peptidyl boronic acid inhibitors for investigations of proteasome-mediated protein degradation was confirmed by the observation that Bz-Phe-boroLeu and Cbz-Leu-Leu-boroLeu pinacol ester inhibited NFĸB activation with IC50 values comparable to their Ki values for purified proteasomes. The latter result supports the view that the chymotrypsin-like activity of proteasomes assayed with suc-Leu-Leu-Val-Tyr-AMC is a critical one for protein degradation in cells.

A facile and high-yield formation of dipyrrin-boronic acid dyads and triads: a light-harvesting system in the visible region based on the efficient energy transfer

2015 ◽  
Vol 13 (9) ◽  
pp. 2574-2581 ◽  
Author(s):  
Masaki Yamamura ◽  
Shinya Yazaki ◽  
Motofumi Seki ◽  
Yasunori Matsui ◽  
Hiroshi Ikeda ◽  
...  
Keyword(s):  
Boronic Acid ◽  
Light Harvesting ◽  
Visible Region ◽  
Boronic Acids ◽  
High Yield ◽  
Artificial Light ◽  
Efficient Energy Transfer ◽  
Efficient Energy ◽  
Harvesting Systems ◽  
Yield Formation

Artificial light-harvesting systems, Ar,O-BODIPY dyads and triads conjugated with a light harvester, were synthesized in high yield by the reaction of an N2O2-type dipyrrin with boronic acids.

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