Purification of Staphylococcus aureus beta-lactamases by using sequential cation-exchange and affinity chromatography

Kernodle, Douglas S.; Zygmunt, Deborah J.; McGraw, Patricia; Chipley, John R.

doi:10.1128/aac.34.11.2177

Cited by 11 publications

(9 citation statements)

References 33 publications

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“…The PC1 ␤-lactamase was partially purified by the procedure of Kernodle et al (14). Briefly, S. aureus PC1 was grown for 16 h at 37°C in 3 liters of modified CY broth (23) containing 0.5 g of methicillin per ml, the cells were removed by centrifugation at 8,000 ϫ g for 10 min, and the supernatant was mixed with 100 ml of P11 cellulose (Whatman) for 1 h at room temperature with shaking.…”

Section: Chemicalsmentioning

confidence: 99%

“…Their levels of transport into bacterial cells, their levels of penicillin-binding protein (PBP) binding, their activities as ␤-lactamase substrates, and the activity of the bound triclosan moiety relative to that of free triclosan against the enoyl reductase target were also determined. (13,17,31) was provided by K. Bush, and S. aureus strains RN4220 and PC1 (14,30,32) were provided by J. Iandolo. E. coli (TEM-1) and its parent strain, E. coli N, have been described previously (16).…”

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confidence: 99%

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Mechanism of Action of NB2001 and NB2030, Novel Antibacterial Agents Activated by β-Lactamases

Stone¹,

Zhang²,

Castillo³

et al. 2004

Antimicrob Agents Chemother

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Two potent antibacterial agents designed to undergo enzyme-catalyzed therapeutic activation were evaluated for their mechanisms of action. The compounds, NB2001 and NB2030, contain a cephalosporin with a thienyl (NB2001) or a tetrazole (NB2030) ring at the C-7 position and are linked to the antibacterial triclosan at the C-3 position. The compounds exploit ␤-lactamases to release triclosan through hydrolysis of the ␤-lactam ring. Like cephalothin, NB2001 and NB2030 were hydrolyzed by class A ␤-lactamases (Escherichia coli TEM-1 and, to a lesser degree, Staphylococcus aureus PC1) and class C ␤-lactamases (Enterobacter cloacae P99 and E. coli AmpC) with comparable catalytic efficiencies (k cat /K m ). They also bound to the penicillin-binding proteins of S. aureus and E. coli, but with reduced affinities relative to that of cephalothin. Accordingly, they produced a cell morphology in E. coli consistent with the toxophore rather than the ␤-lactam being responsible for antibacterial activity. In biochemical assays, they inhibited the triclosan target enoyl reductase (FabI), with 50% inhibitory concentrations being markedly reduced relative to that of free triclosan. The transport of NB2001, NB2030, and triclosan was rapid, with significant accumulation of triclosan in both S. aureus and E. coli. Taken together, the results suggest that NB2001 and NB2030 act primarily as triclosan prodrugs in S. aureus and E. coli.The ubiquitous occurrence of ␤-lactamases in bacteria and their association with clinical resistance to ␤-lactams have allowed strong interest in these enzymes to be sustained (3, 26). ␤-Lactamases are periplasmic in gram-negative bacteria, while they are exocellular in gram-positive bacteria. They are conveniently divided into four classes (classes A, B, C, and D) on the basis of amino acid sequence homologies (Ambler classification) (1, 15). Of these classes, classes A and C are of the greatest clinical significance. ␤-Lactamase inhibitors, such as clavulanic acid and the penicillanic acid sulfones, mostly target class A enzymes; and their use in combination with older compounds has restored the broad-spectrum activities of older compounds, such as amoxicillin (18,22,25,26). Inhibitors that target class C and class B ␤-lactamases have yet to reach clinical development.Exploiting common ␤-lactamases to generate novel antibacterials is a strategy originally used by O'Callaghan et al. (24) and subsequently by Mobashery and Johnston (20) and other investigators (6,10,16). It has been part of NewBiotics' general enzyme-catalyzed therapeutic activation (ECTA) prodrug approach that harnesses unique enzymes in bacteria to achieve selective release of cytotoxic agents from substrate-like molecules (16; M. V.

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Section: Chemicalsmentioning

confidence: 99%

mentioning

confidence: 99%

Mechanism of Action of NB2001 and NB2030, Novel Antibacterial Agents Activated by β-Lactamases

Stone¹,

Zhang²,

Castillo³

et al. 2004

Antimicrob Agents Chemother

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“…Extracellular ,B-lactamase was purified from each of the six S. aureus isolates by sequential cation-exchange and affinity chromatography (7,15 (22). After 2 h, the gels were removed from the soaking solution and left uncovered at room temperature to permit evaporation of excess fluid, until they were within 5% of their original weight.…”

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confidence: 99%

Characterization of four beta-lactamases produced by Staphylococcus aureus

Zygmunt

Stratton

Kernodle

1992

Antimicrob Agents Chemother

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“…The wild-type ␤-lactamase-producing strains and the RN4220 transformants were grown in 5 liters of modified 1% CY medium containing methicillin (0.5 g/ml) or 2-(2Ј-carboxyphenyl)benzoyl-6-aminopenicillanic acid (7.5 M, to induce ␤-lactamase production) at 37ЊC and 150 rpm for 18 h. The extracellular ␤-lactamases were purified by sequential cation-exchange and affinity chromatography (21).…”

mentioning

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Structure-function relationships among wild-type variants of Staphylococcus aureus beta-lactamase: importance of amino acids 128 and 216

1996

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␤-Lactamases inactivate penicillin and cephalosporin antibiotics by hydrolysis of the ␤-lactam ring and are an important mechanism of resistance for many bacterial pathogens. Four wild-type variants of Staphylococcus aureus ␤-lactamase, designated A, B, C, and D, have been identified. Although distinguishable kinetically, they differ in primary structure by only a few amino acids. Using the reported sequences of the A, C, and D enzymes along with crystallographic data about the structure of the type A enzyme to identify amino acid differences located close to the active site, we hypothesized that these differences might explain the kinetic heterogeneity of the wild-type ␤-lactamases. To test this hypothesis, genes encoding the type A, C, and D ␤-lactamases were modified by site-directed mutagenesis, yielding mutant enzymes with single amino acid substitutions. The substitution of asparagine for serine at residue 216 of type A ␤-lactamase resulted in a kinetic profile indistinguishable from that of type C ␤-lactamase, whereas the substitution of serine for asparagine at the same site in the type C enzyme produced a kinetic type A mutant. Similar bidirectional substitutions identified the threonine-to-alanine difference at residue 128 as being responsible for the kinetic differences between the type A and D enzymes. Neither residue 216 nor 128 has previously been shown to be kinetically important among serine-active-site ␤-lactamases.␤-Lactam antibiotics, including the penicillins and cephalosporins, are important agents in the therapy of bacterial infections. However, in some clinical settings the usefulness of these agents has been diminished by the emergence and spread of bacterial strains that produce ␤-lactamase, which hydrolyzes the ␤-lactam ring and inactivates the drug's antimicrobial effect (27). This problem has been demonstrated most dramatically with Staphylococcus aureus. Whereas the vast majority of clinical isolates of S. aureus were highly susceptible to penicillin G at the time of its introduction into clinical use in the early 1940s, the spread of ␤-lactamase-producing, penicillin-resistant strains was so widespread by the late 1940s that penicillin G was no longer a reliable antistaphylococcal agent. Most clinical isolates of S. aureus produce ␤-lactamase (36).Four types of S. aureus ␤-lactamase have been identified by serologic (34, 35) and kinetic (19,20) methods. These variants originally were designated types A, B, C, and D by Richmond (34) and Rosdahl (35). This nomenclature should not be confused with that of the different classes of ␤-lactamases, A through D, that has been used more recently to group the ␤-lactamases of all bacterial species on the basis of active site (serine versus zinc), size, and kinetic characteristics (4). Each of the four recognized types of S. aureus ␤-lactamase (A, B, C, and D) is a class A ␤-lactamase with a serine active site. The mature form of the enzyme has a molecular mass of 30 kDa, contains 257 amino acids, and is excreted extracellularly (1).The type A a...

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Purification of Staphylococcus aureus beta-lactamases by using sequential cation-exchange and affinity chromatography

Cited by 11 publications

References 33 publications

Mechanism of Action of NB2001 and NB2030, Novel Antibacterial Agents Activated by β-Lactamases

Mechanism of Action of NB2001 and NB2030, Novel Antibacterial Agents Activated by β-Lactamases

Characterization of four beta-lactamases produced by Staphylococcus aureus

Structure-function relationships among wild-type variants of Staphylococcus aureus beta-lactamase: importance of amino acids 128 and 216

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