Identification of the active site of <i>Citrobacter freundii</i> β‐lactamase using dansyl‐penicillin

Yamaguchi, Akihito; Adachi, Hideki; Sawai, Tetsuo

doi:10.1016/0014-5793(87)81031-1

Cited by 11 publications

(3 citation statements)

References 14 publications

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“…We selected the cephalosporinase of Citrobacter freundii GN346 as a typical cephalosporinase. Its primary amino acid sequence, consisting of 361 amifno acids, was determined, and the active-site serine was confirmed to be 23,24). We demonstrated the essential role of cephalosporinase Lys-67 in the enzymatic process, which is a conserved residue located three positions downstream of the active-site serine, and found…”

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confidence: 80%

Extension of the substrate spectrum by an amino acid substitution at residue 219 in the Citrobacter freundii cephalosporinase

1990

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The cephalosponnase of Citrobacterfreundii GN346 is a class C P-lactamase, consisting of 361 amino acids and exhibiting the substrate profile of a typical cephalosporinase. On From the viewpoint of clinical medicine, cephalosporinases are more significant ,B-lactamases than the penicilinasetype enzymes, because a high level of cephalosporinase production results in high bacterial resistance to both cephalosporins and penicillins (18). Most cephalosporinases are produced by gram-negative bacteria and are classified as class C P-lactamnases, using Ambler's classification based on the amino acid sequence around the active site (1). Structure-function studies on the active sites of P-lactamases have been mainly devoted to class A P-lactamases such as plasmid-mediated penicillinases (4,(19)(20)(21) and chromosomal penicillinases of gram-positive bacteria (7). Although information regarding the structtre-function of penicillinases is useful for an understanding of cephalosporinases, analysis of cephalosporinase itself is required for elucidation of the characteristics of the enzymes. We selected the cephalosporinase of Citrobacter freundii GN346 as a typical cephalosporinase. Its primary amino acid sequence, consisting of 361 amifno acids, was determined, and the active-site serine was confirmed to be 23,24). We demonstrated the essential role of cephalosporinase Lys-67 in the enzymatic process, which is a conserved residue located three positions downstream of the active-site serine, and found 1-lactamases and penicillin-binding proteins throughout (23).During the course of our subsequent study to investigate, by site-directed mutagenesis, the presence of a hypothetical salt bridge between Lys-67 and an acidic amino acid, we obtained a mutant C. freundii cephalosporinase gene which increased the levels of resistance of Escherichia coli to oxyimino cephalosporins, monobactam, and carbenicillin. This mutation is attributed to the conversion of glutamic acid at residue 219 to lysine, and the mutation is interesting not only as an event affecting the substrate profile, but also as a possible mechanism of bacterial resistance against novel P-lactam antibiotics in gram-negative pathogens.In this paper, we report the properties of the mutant cephalosporinase.* Corresponding author.MATERIALS AND METHODS E. coli strains and plasmids. E. coli TG1 (3), a derivative of K-12, was used for DNA technology and for measuring the antibiotic susceptibility of cells bearing cephalosporinase genes. E. coli AS226-51, an ampD mutant of C600 which also has a deletion mutation in ampC, was used as a host cell for purification of the mutant enzyme to avoid contamination by the ampC J-lactamase of E. coli. Plasmid pCFC-1 is a derivative of pHSG398 into which the wild-type cephalosporinase gene from C. freundii GN346 was inserted (23). pHSG398 carrying the mutant cephalosporinase gene is called pCFC-E219K. The mutant gene was named by using a one-letter amino acid code; i.e., E219K means a mutant gene in which glutamic acid-219 was changed to...

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confidence: 80%

Extension of the substrate spectrum by an amino acid substitution at residue 219 in the Citrobacter freundii cephalosporinase

1990

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“…1). In the case of class C enzymes it seems also wellestablished that, in turnover of penicillins, hydrolysis of the acyl-enzyme intermediate is rate-determining at saturation (kcat = k3) [5][6][7][8]. There has been some general feeling, however, that in class A /,-lactamases acylation of the enzyme is generally the rate-determining step [9][10][11].…”

Section: Introductionmentioning

confidence: 99%

Accumulation of acyl-enzyme intermediates during turnover of penicillins by the class A β-lactamase of Staphylococcus aureus PC1

Pratt

McConnell

Murphy

1988

Biochemical Journal

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The interaction of dansylpenicillin with the class A Staphylococcus aureus PCI beta-lactamase yielded an accumulating intermediate with fluorescence enhanced beyond that of the substrate. Acid quenching of the reaction mixture yielded a denatured enzyme with 1 molar equivalent of dansyl group covalently bound to it. A similar quenching experiment with the PC1 beta-lactamase and [14C]benzylpenicillin yielded an enzyme with 1 molar equivalent of 14C covalently bound. These data indicate that in turnover of S-type penicillins by the PC1 beta-lactamase deacylation is rate-determining. This has not indicate previously been demonstrated for a class A beta-lactamase.

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“…Ten micrograms of membrane proteins was separated by SDSpolyacrylamide gel electrophoresis and then electrically blotted onto nitrocellulose membranes. The TetA proteins were visualized with anti-carboxyl-terminal peptide antiserum and alkaline phosphatase-linked goat anti-rabbit IgG, as described previously (Yamaguchi et al, 1990b).…”

Section: Methodsmentioning

confidence: 99%

Site-Specificity of the Second-Site Suppressor Mutation of the Asp-285 .fwdarw. Asn Mutant of Metal-Tetracycline/H+ Antiporter of Escherichia coli and the Effects of Amino Acid Substitutions at the First and Second Sites

Someya¹,

Niwa²,

Sawai³

et al. 1995

Biochemistry

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The deleterious effect of the mutation of Asp-285 to Asn of the metal-tetracycline/H+ antiporter (TetA) of Escherichia coli is suppressed by the second-site mutation of Ala-220 to an acidic amino acid residue (Yamaguchi, A., O'yauchi, R., Someya, Y., & Sawai, T. (1993) J. Biol. Chem. 268, 26990-26995). In this study, site-specific second-site Glu mutants as to 11 different positions around position 220 were established from the Asp-285-->Asn mutant TetA protein. Among them, only the Ala-220-->Glu mutant completely suppressed the deleterious effect of the Asp-285-->Asn mutation, indicating that position 220 is highly specific for the suppression. Although E. coli cells producing second-site Glu mutants as to positions 213, 216, 217, 218, 219, 221, 222, and 223 of the Asn-285 mutant were as tetracycline sensitive as the host cells without TetA, Gly-224-->Glu and Pro-227-->Glu second-site mutants of the Asn-285 mutant conferred low-level tetracycline resistance, the levels decreasing in this order. These two positions and position 220 are on the same side of putative transmembrane helix VII. The Phe-289-->Asp mutation, which is located at a position one-alpha-helical-turn downstream from Asp-285 in the same putative helix, IX, did not suppress the Asn-285 mutation. The introduction of an acidic residue at the second site was essential for suppression of the Asn-285 mutation because Lys-220 and Gln-220 second-site mutants of the Asn-285 mutant showed very low tetracycline resistance.(ABSTRACT TRUNCATED AT 250 WORDS)

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Identification of the active site of Citrobacter freundii β‐lactamase using dansyl‐penicillin

Cited by 11 publications

References 14 publications

Extension of the substrate spectrum by an amino acid substitution at residue 219 in the Citrobacter freundii cephalosporinase

Extension of the substrate spectrum by an amino acid substitution at residue 219 in the Citrobacter freundii cephalosporinase

Accumulation of acyl-enzyme intermediates during turnover of penicillins by the class A β-lactamase of Staphylococcus aureus PC1

Site-Specificity of the Second-Site Suppressor Mutation of the Asp-285 .fwdarw. Asn Mutant of Metal-Tetracycline/H+ Antiporter of Escherichia coli and the Effects of Amino Acid Substitutions at the First and Second Sites

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