Inactivation of Bacillus cereus .beta.-lactamase I by 6.beta.-bromopenicillanic acid: kinetics

Mj, Loosemore; Sa, Cohen; Rf, Pratt

doi:10.1021/bi00558a016

Cited by 70 publications

(30 citation statements)

References 26 publications

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“…By following this procedure, a colony collection was made, transferred to agar with a 96-prong replicator, and grown for 18 hr. The colonies were "lifted" onto Whatman 541 filter paper for amplification on 250/,g/ml chloramphenicol plates for 24 hr, followed by alkali lysis and neutralization (34). The filters prepared in this way were then prehybridized for 3 hr at 65°C in 6X NaCl/EDTA/Tris, 0.5% Nonidet P-40, and denatured, sonicated salmon sperm DNA at 100 ,g/ml (lx NaCl/EDTA/Tris is 0.15 M NaCl/1 mM EDTA/0.03 M Tris-HC1, pH 8.0).…”

Section: The 16-base Oligodeoxynucleotide 5'-a-a-t-g-a-t-g-a-c-c-t-c-mentioning

confidence: 99%

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Oligonucleotide-directed mutagenesis as a general and powerful method for studies of protein function.

Dalbadie-McFarland¹,

Cohen²,

Riggs³

et al. 1982

Proc. Natl. Acad. Sci. U.S.A.

191

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We have used oligonucleotide-directed mutagenesis to make a specific change in the f-lactamase (EC 3.5.2.6) (ampicillin resistance) gene of the plasmid pBR322. Evidence suggests that the active site for this enzyme may include a serine-threonine dyad (residues 70 and 71). By priming in vitro DNA synthesis with a chemically synthesized 16-base oligodeoxyribonucleotide, we have inverted the Ser-Thr dyad to Thr-Ser and thereby generated a mutant with an ampicillin-sensitive phenotype. This "double-mismatch" method is relatively simple and also very general because detection of mutants is at the level of DNA and involves only colony hybridization. Accordingly, the procedure can be applied to any DNA sequence and does not depend on the phenotype of the mutant.Rational study of the influence of amino acid sequence on the three-dimensional structure and function of a protein would benefit greatly from the ability to change specific residues. When the gene for the protein has been cloned and is expressed in an appropriate vector, site-directed mutagenesis (1, 2) provides a method for creating new proteins whose structural and functional characteristics may be ofpractical value or may offer significant mechanistic insights.Oligonucleotide-directed mutagenesis, the most specific form ofdirected mutagenesis, has been used to produce specific base changes in the single-stranded phage 4X174 (3, 4). The method ( Fig. 1) involves priming in vitro DNA synthesis with a chemically synthesized oligodeoxynucleotide that carries at least a single base mismatch with the complementary strand of the "wild-type" DNA. Because DNA polymerases require a double-stranded segment for initiation of DNA replication, the synthetic oligodeoxynucleotide primes DNA synthesis and is, itself, incorporated into the resulting heteroduplex molecule. After molecular cloning, semiconservative in vivo replication of this heteroduplex gives rise to homoduplexes whose sequences are either that of the original wild-type DNA or that of the synthetic oligodeoxynucleotide. With single-stranded circular DNAs, priming with oligodeoxynucleotides has been used to cause transitions, transversions, and deletions, in some cases very efficiently (5-11). Wallace et aL (12, 13) have extended the technique to double-stranded circular DNAs, and even though the frequency of directed mutagenesis may be lower, screening of colonies with the 32P-labeled synthetic oligodeoxynucleotide allows easy detection of the desired mutant (12, 13).The particular virtue of oligonucleotide-directed mutagenesis for structure-function studies lies in allowing one to produce mutant proteins with very specific changes in particular residues, which, for example, may be directly involved in catalysis or in determining substrate specificity. In this way, one can test ideas about the roles ofparticular amino acid side chains. Other procedures are valuable for producing random or less specific changes as, for example, in the creation of a mutant of f3lac-tamase (EC 3.5.2.6) with enhanced act...

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Section: The 16-base Oligodeoxynucleotide 5'-a-a-t-g-a-t-g-a-c-c-t-c-mentioning

confidence: 99%

“…A serine residue probably participates in catalysis (21)(22)(23)(24)(25)(26) and is part of a conserved Ser-Thr dyad (26). [These residues have been numbered and Thr-71 (26).]…”

mentioning

confidence: 99%

Oligonucleotide-directed mutagenesis as a general and powerful method for studies of protein function.

Dalbadie-McFarland¹,

Cohen²,

Riggs³

et al. 1982

Proc. Natl. Acad. Sci. U.S.A.

191

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“…6-β-Iodopenicillanates and 6-β-bromopenicillanates are powerful inhibitors of β-lactamases 10–14. Inactivation of the class A β-lactamase from Bacillus cereus by BIP was shown to be accompanied by the formation of a new chromophore 15,16 and analysis of the absorption, circular dichroic and NMR spectra of the protein-bound chromophore or the isolated chromophore gave strong evidence that BIP covalently binds to the enzyme and undergoes a rearrangement of the penicilloyl-enzyme intermediate, possibly via ring opening of the thiazolidine and reaction of the thiolate anion thereby formed 17.…”

Section: Introductionmentioning

confidence: 99%

Structural Basis of the Inhibition of Class A β-Lactamases and Penicillin-Binding Proteins by 6-β-Iodopenicillanate

et al. 2009

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6-β-Halogenopenicillanates are powerful irreversible inhibitors of various β-lactamases and penicillin binding proteins. Upon acylation of these enzymes, the inhibitors are thought to undergo a structural rearrangement associated with the departure of the iodide and formation of a dihydrothiazine ring, but, to date, no structural evidence has proven this. 6-β-Iodopenicillanic (BIP) is here shown to be an active antibiotic against various bacterial strains and an effective inhibitor of the class A β-lactamase of Bacillus subtilis BS3 (BS3) and the D,D-peptidase of Actinomadura R39 (R39). Crystals of BS3 and of R39 were soaked with a solution of BIP and their structures solved at 1.65 Å and 2.2 Å, respectively. The β-lactam and the thiazolidine rings of BIP are indeed found to be fused into a dihydrothiazine ring that can adopt two stable conformations at these active sites. The rearranged BIP is observed in one conformation in the BS3 active site and in two monomers of the asymmetric unit of R39, and is observed in the other conformation in the other two monomers of the asymmetric unit of R39. The BS3 structure reveals a new mode of carboxylate interaction with a class A β-lactamase active site that should be of interest in future inhibitor design.

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“…With respect to the branched mechanism of inhibition, the features of 3-lactamase inactivation by 7ac-formylamino compounds resembled those by P-lactamase inhibitors such as 3-bromopenicillanic acid (7), clavulanic acid (6), penam sulfones (4,5), and carbapenems (2). Turnover numbers of 7cc-formylamino compounds were smaller than those of clavulanic acid (115 for TEM penicillinase [6] and 70 for cephalosporinase of P. vulgaris [23]) and of sulbactam (5,200 for TEM penicillinase and 80 for cephalosporinase of C. freundii [24]) and larger than that of asparenomycin A (1.8 for cephalosporinase of C. freundii [8]).…”

Section: Discussionmentioning

confidence: 99%

7 alpha-formylamino substituent confers beta-lactamase-inactivating potency on 1-oxacephalosporins

Murakami¹,

Doi²,

Yoshida³

1986

Antimicrob Agents Chemother

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acid (Fl) and 7ft-formylamino-7 -1 -2-[(4-ethyl-2,3-dioxopiperazine-1-yl)carbonylamino]-2-phenylacetamido}-3-[( 1-methyl-1H-tetrazol-5-yl)thiomethyl]-1-oxa-3-cephem-4-carboxylic acid (F2) were stable against penicillinases and, moreover, inactivated cephalosporinases of Pseudomonas aeruginosa, Citrobacterfreundii, and Enterobacter cloacae.Extensive studies of the inactivation of cephalosporinase of P. aeruginosa showed that it resulted from the formation of a transiently stable enzyme-compound complex. The 7a-formylamino substituent was involved in the enzyme inactivation, because 7a-methoxy congeners did not inactivate the enzyme. The number of compound molecules required for inhibition of an enzyme molecule was found to be 36 for Fl and 5.5 for F2, which suggests that the pathway to the complex formation branched off the hydrolysis pathway. Half-lives of the complexes were 400 min for Fl and 260 min for F2. 7a-Formylamino compounds Fl and F2 had antibacterial activities similar to those of 7fi-methoxy congeners against ,-lactamase-producing gram-negative bacteria, whereas they were less active against non-1-lactamase-producing strains of Escherichia coli and Staphylococcus aureus. The conclusion was that the 7a-formylamino substituent conferred the ability to inactivate cephalosporinase on the 1-oxacephalosporins tested, without much impairment of their antibacterial activity.,-Lactamase catalyzes cleavage of the amide bond of the P-lactam ring of ,-lactam antibiotics, rendering the antibiotics inactive, and r-lactamase production is the main cause of bacterial resistance to P-lactam antibiotics (16,21). Thus, the introduction of substituents which stabilize 3-lactam compounds against r-lactamases or inhibit the enzyme is very important for the development of antibiotics having high activities against f3-lactamase-producing bacteria.Cephalosporins containing the 7oc-methoxy substituent (cephamycins) were found in nature in the early 1970s (12,20). This substituent stabilizes cephamycins against 1-lactamases (3). Since then, 7ox-substituted cephalosporins and 6(-substituted penicillins have become a very important group of 1-lactam antibiotics. In screening programs, many 7(-and 7,B-substituted 1-oxacephalosporins have been synthesized, and their biological activities were tested in our laboratories (13). Recent discoveries of naturally occurring 7ot-formylaminocephalosporins in our and other laboratories (18,19,22) have stimulated the synthesis of two 7a-formylamino-1-oxacephalosporins in our laboratories. We examined the interaction of these two compounds with f-lactamases and found that the 7ot-formylamino-1-oxacephalosporins inactivated cephalosporinase by forming a transiently stable complex with the enzyme, but 7ot-methoxy congeners did not. (University of Manchester, Manchester, England), and E. coli ML1410 carrying R-plasmid RGN238 and Klebsiella pneumoniae GN69 were obtained from T. Sawai (Chiba University, Chibashi, Japan). Other strains were clinical isolates stocked in our laboratories. Preparation ...

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Inactivation of Bacillus cereus .beta.-lactamase I by 6.beta.-bromopenicillanic acid: kinetics

Cited by 70 publications

References 26 publications

Oligonucleotide-directed mutagenesis as a general and powerful method for studies of protein function.

Oligonucleotide-directed mutagenesis as a general and powerful method for studies of protein function.

Structural Basis of the Inhibition of Class A β-Lactamases and Penicillin-Binding Proteins by 6-β-Iodopenicillanate

7 alpha-formylamino substituent confers beta-lactamase-inactivating potency on 1-oxacephalosporins

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