scholarly journals Mechanism of acyl transfer by the class A serine β-lactamase of Streptomyces albus G

1991 ◽  
Vol 279 (1) ◽  
pp. 213-221 ◽  
Author(s):  
J Lamotte-Brasseur ◽  
G Dive ◽  
O Dideberg ◽  
P Charlier ◽  
J M Frère ◽  
...  
Keyword(s):  
Carbon Atom ◽  
Amide Bond ◽  
Carbonyl Carbon Atom ◽  
Cephalosporin C ◽  
Beta Lactamase ◽  
Beta Lactam ◽  
Carbonyl Carbon ◽  
Class A ◽  
Streptomyces Albus ◽  
Hydrolysis Of

Optimization by energy minimization of stable complexes occurring along the pathway of hydrolysis of benzylpenicillin and cephalosporin C by the Streptomyces albus G beta-lactamase has highlighted a proton shuttle that may explain the catalytic mechanism of the beta-lactamases of class A. Five residues, S70, S130, N132, T235 and A237, are involved in ligand binding. The gamma-OH group of T235 and, in the case of benzylpenicillin, the gamma-OH group of S130 interact with the carboxylate group, on one side of the ligand molecule. The side-chain NH2 group of N132 and the carbonyl backbone of A237 interact with the exocyclic CONH amide bond, on the other side of the ligand. The backbone NH groups of S70 and A237 polarize the carbonyl group of the scissile beta-lactam amide bond. Four residues, S70, K73, S130 and E166, and two water molecules, W1 and W2, perform hydrolysis of the bound beta-lactam compound. E166, via W1, abstracts the proton from the gamma-OH group of S70. While losing its proton, the O-gamma atom of S70 attacks the carbonyl carbon atom of the beta-lactam ring and, concomitantly, the proton is delivered back to the adjacent nitrogen atom via W2, K73 and S130, thus achieving formation of the acyl-enzyme. Subsequently, E166 abstracts a proton from W1. While losing its proton, W1 attacks the carbonyl carbon atom of the S70 ester-linked acyl-enzyme and, concomitantly, re-entry of a water molecule W'1 replacing W1 allows E166 to deliver the proton back to the same carbonyl carbon atom, thus achieving hydrolysis of the beta-lactam compound and enzyme recovery. The model well explains the differences found in the kcat. values for hydrolysis of benzylpenicillin and cephalosporin C by the Streptomyces albus G beta-lactamase. It also explains the effects caused by site-directed mutagenesis of the Bacillus cereus beta-lactamase I [Gibson, Christensen & Waley (1990) Biochem J. 272, 613-619].

scholarly journals Interaction of β-iodopenicillanate with the β-lactamases of Streptomyces albus G and Actinomadura R39

1982 ◽  
Vol 207 (3) ◽  
pp. 437-444 ◽  
Author(s):  
J M Frère ◽  
C Dormans ◽  
C Duyckaerts ◽  
J De Graeve
Keyword(s):  
Bacillus Cereus ◽  
Absorption Maximum ◽  
Amide Bond ◽  
Beta Lactamase ◽  
Beta Lactam ◽  
Beta Lactamases ◽  
Streptomyces Albus ◽  
Hydrolysis Of

The beta-lactamases of Streptomyces albus G and Actinomadura R39 are inactivated by beta-iodopenicillanate. However, in contrast with the beta-lactamase I from Bacillus cereus, they also efficiently catalyse the hydrolysis of the inactivator; with the S. albus G enzyme, kcat. is larger than 25s-1 and the number of turnovers before inactivation is 515. With the A. R39 enzyme, kcat. is larger than 50s-1 and the number of turnovers before inactivation is 80. After hydrolysis of the beta-lactam amide bond, the product rearranges into 2.3-dihydro-2,2-dimethyl-1,4-thiazine-3,6-dicarboxylate, which exhibits an absorption maximum at 305 nm.

Characteristics of Aerobacter beta-lactamase

1968 ◽  
Vol 14 (2) ◽  
pp. 139-145 ◽  
Author(s):  
M. Goldner ◽  
D. G. Glass ◽  
P. C. Fleming
Keyword(s):  
Reaction Rates ◽  
Penicillin G ◽  
Cephalosporin C ◽  
Beta Lactamase ◽  
Beta Lactam ◽  
Lactam Ring ◽  
Rate Of Hydrolysis ◽  
High Concentrations ◽  
Hydrolysis Of ◽  
Pseudomonas Pyocyanea

In this investigation, Aerobacter cloacae is shown to inactivate cephalosporin by hydrolysis of its beta-lactam ring. This was demonstrated by iodine absorption and infrared absorption spectra.The values of the Michaelis constant obtained with cephalosporin C and deacetyl cephalosporin C indicate a great affinity of the Aerobacter's beta-lactamase for its substrate. The enzyme was most active at pH 7.0 and 37 C. Aqueous washings of the Aerobacter cells were a potent source of enzyme.The beta-lactamase of A. cloacae was active on both cephalosporin and penicillin. A higher rate of hydrolysis was observed with cephalosporin C and deacetyl cephalosporin C than with cephalothin and cephaloridine. The ratio of reaction rates on cephalosporin C to that on penicillin G was consistently of the order of 100 to 1. The activity on V, N, and especially the semisynthetic penicillins was also low.The A. cloacae enzyme was easily demonstrable in large amount without added inducer. By contrast, the activity of the beta-lactamase from Pseudomonas pyocyanea cannot be detected unless high concentrations of inducer are used.

scholarly journals Interaction of clavulanate with the β-lactamases of Streptomyces albus G and Actinomadura R39

1982 ◽  
Vol 207 (3) ◽  
pp. 429-436 ◽  
Author(s):  
J M Frère ◽  
C Dormans ◽  
V M Lenzini ◽  
C Duyckaerts
Keyword(s):  
Kinetic Models ◽  
Reaction Scheme ◽  
Beta Lactamase ◽  
Beta Lactam ◽  
General Reaction ◽  
Beta Lactamases ◽  
Complete Inactivation ◽  
Streptomyces Albus ◽  
Hydrolysis Of

The reactions of beta-lactamases of Actinomadura R39 and Streptomyces albus G with clavulanate proceed along branched pathways. Both enzymes perform the hydrolysis of this beta-lactam with rather high efficiencies (kcat. = 18s-1 and 52s-1 respectively). If large clavulanate/enzyme ratios are used, complete inactivation of the enzymes is observed. At lower ratios, inactivation is only partial. Irreversible inactivation occurs after 400 and 20000 turnovers for the A. R39 and S. albus G enzymes respectively. With the A. R39 beta-lactamase, a transiently inhibited complex is also formed that remains undetectable with the S. albus G beta-lactamase. Kinetic models are presented and studied for the interaction between clavulanate and both enzymes. A tentative general reaction scheme is also discussed.

scholarly journals A metallo-beta-lactamase with both beta-lactamase and ribonuclease activity is linked with traduction in giant viruses

2019 ◽  
Author(s):  
Philippe Colson ◽  
Lucile Pinault ◽  
Said Azza ◽  
Nicholas Armstrong ◽  
Eric Chabriere ◽  
...  
Keyword(s):  
Acanthamoeba Castellanii ◽  
Deep Ocean ◽  
Penicillin G ◽  
Beta Lactamase ◽  
Beta Lactam ◽  
Strong Activity ◽  
Beta Lactam Antibiotics ◽  
Double Stranded Dna ◽  
Giant Viruses ◽  
Hydrolysis Of

ABSTRACTEnzymatic proteins with a metallo-beta-lactamase (MBL) fold have been essentially studied in bacteria for their activity on beta-lactam antibiotics. However, the MBL fold is ancient and highly conserved, and these proteins are capable of cleaving a broad range of substrates. It has recently been shown that MBLs are present in a wide array of cellular organisms, including eukaryotes and archaea. We show here that Tupanvirus deep ocean, a giant virus, also encodes a protein with a MBL fold. Phylogeny showed its clustering with transfer ribonucleases (RNases) and the presence of orthologs in other giant viruses, mainly those harboring the largest sets of translation components. In addition, it suggests an ancient origin for these genes and a transfer between giant viruses and Acanthamoeba spp., a host of many giant viruses. Biologically, after its expression in Escherichia coli, the tupanvirus protein was found to hydrolyse nitrocefin, a chromogenic beta-lactam. We also observed an hydrolysis of penicillin G (10 μg/mL) and detected the metabolite of penicillin G hydrolysis, benzylpenilloic acid. This was inhibited by sulbactam, a beta-lactamase inhibitor. In addition, we tested the degradation of single-stranded DNA, double-stranded DNA, and RNAs, and observed a strong activity on RNAs from seven bacteria with G+C varying from 42% to 67%, and from Acanthamoeba castellanii, the tupanvirus host. This was not inhibited by sulbactam or ceftriaxone. RNase activity was estimated to be 0.45±0.15 mU/mg using a fluorescence-based assay. Our results still broaden the range of hosts of MBL fold proteins and demonstrate that such protein can have dual beta-lactamase/nuclease activities. We suggest that they should be annotated according to this finding to avoid further confusion.

Argon Matrix Infrared Spectroscopic Evidence for the Generation of Pentatetraenone by Flash Pyrolysis of Suitable Precursors

1988 ◽  
Vol 41 (2) ◽  
pp. 225 ◽  
Author(s):  
RFC Brown ◽  
KJ Coulston ◽  
FW Eastwood ◽  
MJ Irvine ◽  
ADE Pullin
Keyword(s):  
Carbon Atom ◽  
Infrared Spectra ◽  
Spectral Region ◽  
Narrow Band ◽  
Broad Band ◽  
Carbonyl Carbon Atom ◽  
Carbonyl Carbon ◽  
Argon Matrix ◽  
Spectroscopic Evidence ◽  
Argon Mixture

Five compounds were investigated as precursors for the pyrolytic generation of pentatetraenone, H2C=C=C=C=C=O. These were (1)-(4): 3- ethenylidenebicyclo [2.2.1]hept-5-ene with the following 2,2 substituents : H, COOCOCF3 (1); H, 13COOCOCF3 (1′); (COOCOCF3)2 (2); (COO)2C(CH3)(OCH3) (3); (COO)2Si(CH3)2 (4) and 5-(3′- methylenebicyclo [2.2.1]hept-5′-en-2′-ylidene)-2,2-dimethyl-1,3-dioxan-4,6-dione (5). The five precursors were pyrolysed in a stream of argon at temperatures in the range 350-725°C and the pyrolysate -argon mixture condensed on a CsI plate at c. 10 K. Infrared spectra were obtained between 4000 and 250 cm-1. All five precursors gave two strong bands in the spectral region 2070-2250 cm-1, possibly attributable to pentatetraenone. At lower pyrolytic temperatures the more intense of the two bands was a broad band centred at c. 2128 cm-1 [precursors (1)- (4)] or at c. 2094 cm-1 [precursor (5)]. At higher pyrolytic temperatures these bands were diminished in intensity and replaced by a narrow band at 2207 cm-1 for all five precursors. Bands due to the expected other products for each pyrolysis reaction to form pentatetraenone were observed. H2C413CO ( pentatetraenone substituted by 13C at the carbonyl carbon atom) was prepared by pyrolysis of precursor (1′). We assign the broad bands at c. 2128 cm-1 [precursors (1)-(4)] and at c. 2094 [precursor (5)] to incompletely pyrolysed precursor in which cyclopentadiene has been retained but decomposition in the rest of the molecule has resulted in formation of a =C=C=O group. Bands at 2207, 2068 and 1726 cm-1 we assign to v2-v4 of pentatetraenone. Corresponding bands at 2168, 2056 and 1720 cm-1 are observed in the spectrum of H2C413CO.

scholarly journals Streptomyces albus G serine β-lactamase. Probing of the catalytic mechanism via molecular modelling of mutant enzymes

1992 ◽  
Vol 282 (1) ◽  
pp. 189-195 ◽  
Author(s):  
J Lamotte-Brasseur ◽  
F Jacob-Dubuisson ◽  
G Dive ◽  
J M Frère ◽  
J M Ghuysen
Keyword(s):  
Hydrogen Bonding ◽  
Active Site ◽  
Catalytic Mechanism ◽  
Site Directed Mutagenesis ◽  
Network Configuration ◽  
Beta Lactam ◽  
Beta Lactamases ◽  
Class A ◽  
Streptomyces Albus ◽  
Hydrogen Bonding Network

In previous studies, several amino acids of the active site of class A beta-lactamases have been modified by site-directed mutagenesis. On the basis of the catalytic mechanism proposed for the Streptomyces albus G beta-lactamase [Lamotte-Brasseur, Dive, Dideberg, Charlier, Frère & Ghuysen (1991) Biochem. J. 279, 213-221], the influence that these mutations exert on the hydrogen-bonding network of the active site has been analysed by molecular mechanics. The results satisfactorily explain the effects of the mutations on the kinetic parameters of the enzyme's activity towards a set of substrates. The present study also shows that, upon binding a properly structured beta-lactam compound, the impaired cavity of a mutant enzyme can readopt a functional hydrogen-bonding-network configuration.

Aril Azines. II. Hydrogenation of p-Tolil Monoazine

1975 ◽  
Vol 53 (5) ◽  
pp. 748-752 ◽  
Author(s):  
Peter Yates ◽  
E. M. Levi
Keyword(s):  
Hydrogen Atom ◽  
Carbon Atom ◽  
Sodium Borohydride ◽  
Sodium Methoxide ◽  
Major Product ◽  
Lead Tetraacetate ◽  
Ring Closure ◽  
Carbonyl Carbon Atom ◽  
Carbonyl Carbon ◽  
Independent Synthesis

Hydrogenation of p-tolil monoazine (1b) over palladium-on-charcoal gives as the major product 4,5-dihydro-5-(p-toluyl)-3,4,5-tri-(p-tolyl)-1H-pyrazol-4-ol (2b), which has previously been obtained by treatment of 1b with sodium methoxide. Several minor products are formed, which include p-tolualdehyde, p-toluic acid, and p-toluamide, p-tolunitrile, p-tolualazine, and 3,4,5-tri-(p-tolyl)-4H-pyrazo-4-ol (9). The structure of the last compound, which is also formed on reduction of 1b with sodium borohydride, was established by its independent synthesis from 1,2,3-tri-(p-tolyl)-1,3-propanedione by oxidation with lead tetraacetate followed by treatment with hydrazine. It is suggested that 2b arises via reduction of a C=N bond of 1b and aldol ring closure. The minor hydrogenation products are of interest in that their formation involves C—C hydrogenolysis; it is suggested that this is initiated by addition of a hydrogen atom to a carbonyl carbon atom of 1b.

scholarly journals Substrate-induced inactivation of the OXA2 β-lactamase

1993 ◽  
Vol 295 (3) ◽  
pp. 871-878 ◽  
Author(s):  
P Ledent ◽  
J M Frère
Keyword(s):  
Competitive Inhibition ◽  
Hydrolysis Product ◽  
Beta Lactamase ◽  
Beta Lactam ◽  
Hydrolysis Time ◽  
Beta Lactam Antibiotics ◽  
Class D ◽  
Time Courses ◽  
Burst Kinetics ◽  
Hydrolysis Of

The hydrolysis time courses of 22 beta-lactam antibiotics by the class D OXA2 beta-lactamase were studied. Among these, only three appeared to correspond to the integrated Henri-Michaelis equation. ‘Burst’ kinetics, implying branched pathways, were observed with most penicillins, cephalosporins and with flomoxef and imipenem. Kinetic parameters characteristic of the different phases of the hydrolysis were determined for some substrates. Mechanisms generally accepted to explain such reversible partial inactivations involving branches at either the free enzyme or the acyl-enzyme were inadequate to explain the enzyme behaviour. The hydrolysis of imipenem was characterized by the occurrence of two ‘bursts’, and that of nitrocefin by a partial substrate-induced inactivation complicated by a competitive inhibition by the hydrolysis product.

scholarly journals ACI-1 class A beta-lactamase is widespread across human gut microbiomes due to transposons harboured by tailed prophages

2017 ◽  
Author(s):  
Chris M Rands ◽  
Elizaveta V Starikova ◽  
Harald Brüssow ◽  
Evgenia V Kriventseva ◽  
Vadim M Govorun ◽  
...  
Keyword(s):  
Antibiotic Resistance ◽  
Antibiotic Resistance Genes ◽  
Mobile Element ◽  
Antibiotics Resistance ◽  
Beta Lactamase ◽  
Mobile Dna ◽  
Beta Lactam ◽  
Human Gut ◽  
Class A ◽  
The Usa

AbstractAntibiotic resistance is increasing among pathogens at unprecedented rates and the human body contains a large pool of antibiotic resistance genes that can be spread among bacteria by mobile genetic elements. Acidaminococcus intestini, a bacterium found in the human gut that belongs to the class of Negativicutes, is the first gram-negative coccus shown to be resistant to beta-lactam antibiotics. Resistance is conferred by aci1, a gene encoding the ACI-1 class A beta-lactamase, but the evolutionary history of aci1 and its distribution across other Negativicutes and in the human gut microbiota remains obscure. We discovered that ACI-1 proteins are phylogenetically distinct from class A beta-lactamases of gram-positive Firmicutes and that the aci1 gene occurs in bacteria scattered across the Negativicutes clade, suggesting possible mobilization. In the reference A. intestini RyC-MR95 strain, we found that aci1 is surrounded by mobile DNA, transposon derived sequences directly flank aci1 and are likely the vehicle for its mobility. These transposon sequences reside within a prophage context consisting of two likely degraded tailed prophages, the first prophages to be characterised in A. intestini. We found aci1 in at least 56 (4.4%) out of 1,267 human gut metagenome samples, mostly hosted within A. intestini, and, where could be determined, mostly within a similar constellation of mobile elements to that found in the reference A. intestini genome. These human samples are from individuals in Europe, China and the USA, showing that aci1 is widely distributed globally. Additionally, we examined the nine different Negativicute genome assemblies that contain aci1, and found that only two of these strains show a similar mobile element context around aci1 to the reference A. intestini with transposons adjacent to a tailed prophage. However, in all nine cases aci1 is flanked by transposon derived sequences, and these sequences are diverse, suggesting the activity and degradation of multiple transposons. Overall, we show that ACI-1 proteins form a distinct class A beta lactamase family, and that the aci1 gene is present in human guts worldwide within Negativicute bacterial hosts, due to transposons, sometimes inserted into tailed prophages.

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