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Identification of an essential glutamic acid residue in beta-lactamase II from Bacillus cereus.

ABSTRACT: Beta-Lactamase II from Bacillus cereus was readily inactivated by incubation at pH 4.75 with a water-soluble carbodiimide plus a suitable nucleophile. In the early stages of the reaction, 1 equivalent of nucleophile was incorporated/equivalent of enzyme, whereas during the later stages a second equivalent of nucleophile was also incorporated. This latter process correlated with the blocking of the enzyme's single thiol group. Enzyme inactivated in the presence of the coloured nucleophile N-(2,4-dinitrophenyl)ethylenediamine was fragmented by pepsin digestion, and coloured peptides were isolated by gel filtration and h.p.l.c. Two major peptides, representing 52% of the incorporated label, were isolated and sequenced. Both peptides contained the incorporated label on glutamic acid-37, and it is concluded that this latter residue represents a catalytically essential carboxylic residue in beta-lactamase II.

SUBMITTER: Little C

PROVIDER: S-EPMC1153048 | biostudies-other | 1986 Jan

REPOSITORIES: biostudies-other

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Identification of beta-lactamase in antibiotic-resistant Bacillus cereus spores.

Project description:Beta-lactamase type I is reported for the first time to occur in the sporulated form in a penicillin-resistant Bacillus species. The enzyme was readily characterized from the B. cereus 5/B line (ATCC 13061) by mass spectrometry and two-dimensional gel electrophoresis.

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Comparison of beta-lactamase II from Bacillus cereus 569/H/9 with a beta-lactamase from Bacillus cereus 5/B/6.

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The composition of beta-lactamase I and beta-lactamase II from Bacillus cereus 569-H.

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Separation, purification and properties of beta-lactamase I and beta-lactamase II from Bacillus cereus 569/H/9.

Project description:1. A procedure was devised which is suitable for the isolation of beta-lactamase I and beta-lactamase II from Bacillus cereus 569/H/9 on a large scale. After adsorption on to Celite both enzymes were eluted in good yield and separated by chromatography on Sephadex CM-50. 2. beta-Lactamase I was separated into three main components by isoelectric focusing and into two components by chromatography. 3. The Zn(2+)-requiring beta-lactamase II obtained by this procedure had a lower molecular weight (22000) than beta-lactamase I (28000) and also differed from the latter in containing one cysteine residue. 4. The beta-lactamase II contained no carbohydrate, but showed the thermostability of the enzyme isolated earlier as a protein-carbohydrate complex. 5. Amino acid analyses and tryptic-digest ;maps' indicate that some degree of homology between beta-lactamase I and beta-lactamase II is possible, but that beta-lactamase I is not composed of the entire sequence of beta-lactamase II together with an additional peptide fragment. 6. A 6-methylpenicillin and a 7-methylcephalosporin showed much lower affinities for both enzymes than did penicillins and cephalosporins themselves.

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A thiono-beta-lactam substrate for the beta-lactamase II of Bacillus cereus. Evidence for direct interaction between the essential metal ion and substrate.

Project description:An 8-thionocephalosporin was shown to be a substrate of the beta-lactamase II of Bacillus cereus, a zinc metalloenzyme. Although it is a poorer substrate, as judged by the Kcat./Km parameter, than the corresponding 8-oxocephalosporin, the discrimination against sulphur decreased when the bivalent metal ion in the enzyme active site was varied in the order Mn2+ (the manganese enzyme catalysed the hydrolysis of the oxo compound but not that of the thiono compound), Zn2+, Co2+ and Cd2+. This result is taken as evidence for kinetically significant direct contact between the active-site metal ion of beta-lactamase II and the beta-lactam carbonyl heteroatom. No evidence was obtained, however, for accumulation of an intermediate with such co-ordination present.

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Engineered mononuclear variants in Bacillus cereus metallo-beta-lactamase BcII are inactive.

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An X-ray-crystallographic study of beta-lactamase II from Bacillus cereus at 0.35 nm resolution.

Project description:Crystals of beta-lactamase II (EC 3.5.2.6., 'penicillinase') from Bacillus cereus were grown with Cd(II) in place of the natural Zn(II) cofactor and stabilized by cross-linking with glutaraldehyde. Their space group is C2, the cell dimensions are a = 5.44 nm, b = 6.38 nm, c = 7.09 nm and beta = 93.6 degrees, and there is one molecule in the asymmetric unit. Diffraction data were collected from cross-linked crystals of the Cd(II)-enzyme, the apoenzyme and six heavy-atom derivatives. The electron-density map calculated at 0.35 nm resolution reveals the essential Cd(II) ion surrounded by three histidine residues and one cysteine residue. The position of a glutamic acid residue, modification of which destroys activity [Little, Emanuel, Gagnon & Waley (1986) Biochem. J. 233, 465-469], suggests the probable location of the active site of the enzyme. Two minor Cd(II) sites not essential for activity were also located. The structure of the apoenzyme at this resolution appears to differ from that of the Cd(II)-enzyme only in the orientation of two of the histidine residues and the cysteine residue that surround the metal ion.

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beta-lactamase I from Bacillus cereus. Structure and site-directed mutagenesis.

Project description:The sequence of the gene for beta-lactamase I from Bacillus cereus 569/H has been redetermined. Oligonucleotide-directed mutagenesis has been carried out, and the effects of the changes on the ampicillin-resistance of Escherichia coli TG1 expressing the mutant genes have been studied. Lysine-73, close to the active-site serine-70 and a highly-conserved residue, has been converted into arginine. This change had a large effect on activity, but did not abolish it. An even larger effect was found in the mutant in which glutamate-166 had been converted into glutamine; this had little or no activity. On the other hand, the conversion of glutamate-168 into aspartate gave fully active enzyme. Glutamate-166 is an invariant residue, but glutamate-168 is not. Alanine-123 has been replaced by cysteine, to give active enzyme; this change forms part of the plan to introduce a disulphide bond into the enzyme.

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The partial amino acid sequence of the extracellular beta-lactamase I of Bacillus cereus 569/H.

Project description:The chemical structure of the extracellular beta-lactamase I of Bacillus cereus 569/H was investigated. Three electrophoretically homogenous charge variants of this enzyme were isolated and amino acid analysis of each revealed no significant differences. However, a degree of N-terminal heterogeneity was found by direct end-group modification of the protein and also on alignment of peptides from tryptic and chymotryptic digestion. The N-terminal heterogeneity observed was great enough to explain the production of the beta-lactamase I isoenzymes which are probably produced by postsynthesis modification of a single gene product. Over 80% of the amino acid sequence of beta-lactamase I was determined by the detailed analysis of peptides derived from tryptic, chymotryptic and thermolytic digests. Five polypeptide fragments were constructed from these data and aligned by comparison with the known amino acid sequences of the penicillinases produced by Bacillus licheniformis and Staphylococcus aureus (Ambler & Meadway, 1969). About 60% of the proposed sequence was identical with that of B. licheniformis penicillinase, whereas the S. aureus enzyme had only about 40% of its residues in common with beta-lactamase I. These results are discussed with reference to the possible evolutionary relationships existing between known beta-lactamases. Detailed evidence for the amino acid sequence proposed has been deposited as Supplementary Publication SUP 50044 (27 pages) at the British Library (Lending Division), Boston Spa, Wetherby, W. Yorks. LS23 7BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1975), 145, 5.

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Irreversible inactivation of beta-lactamase I from Bacillus cereus by chlorinated 6-spiroepoxypenicillins.

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