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J R Knox

Publications and source records attributed to J R Knox.

At least 19 recordsLinked to original sources
PubMed1992

Site-saturation mutagenesis and three-dimensional modelling of ROB-1 define a substrate binding role of Ser130 in class A beta-lactamases.

Site-saturation mutagenesis was performed on the class A ROB-1 beta-lactamase at conserved Ser130, which is centrally located in the antibiotic binding site where it can participate in both protein-protein and protein-substrate hydrogen bonding. Mutation Thr130 gave a beta-lactamase hydrolysing penicillins and cephalosporins but which showed a 3-fold lower affinity (Km) for ampicillin and cephalexin, and a 30-fold lower hydrolytic (Vmax) activity for ampicillin. In contrast, the hydrolytic activity for cephalexin was similar to the wild-type for the Thr130 mutation. Mutation Gly130 gave a beta-lactamase hydrolysing only penicillins with an affinity and hydrolysis activity for these compounds approximately 15-fold lower than the wild-type, but no detectable activity against cephalosporins. Mutation Ala130 produced an enzyme capable of hydrolysing penicillins only at a low rate. Modelling the ROB-1 active site was done from the refined 2 A X-ray structure of the homologous Bacillus licheniformis beta-lactamase. Ampicillin and cephalexin were docked into the active site and were energy minimized with the CVFF empirical force field. Dockings were stable only when Ser70 was made anionic and Glu166 was made neutral. Interaction energies and distances were calculated for fully hydrated pre-acylation complexes with the Ser, Thr, Gly and Ala130 enzymes. The catalytic data from all mutations and the computed interactions from modelling confirmed that the Ser130 has a structural as well as a functional role in binding and hydrolysis of penicillins. This highly conserved residue also plays a substrate specificity role by hydrogen binding the carboxylic acid group of cephalosporins more tightly than penicillins.

Ampicillin↗
PubMed1991

Beta-lactamase of Bacillus licheniformis 749/C. Refinement at 2 A resolution and analysis of hydration.

The crystallographic and molecular structure of the class A beta-lactamase (penicillinase) of Bacillus licheniformis 749/C has been refined with X-ray diffraction data to 2 A resolution. For the 27,330 data with F greater than or equal to 3 sigma(F), the R factor is 0.15; for all 30,090 data, R is 0.16. The estimated co-ordinate error is 0.15 A. In the final model, the deviation of covalent bonds and angles from ideality is 0.012 A and 2.2 degrees, respectively. The model includes two molecules of 29,500 daltons each in the asymmetric unit of space group P2(1), 484 water molecules and two tetrahedral buffer anions. Overlay of the two protein molecules results in a root-mean-square difference of 0.17 A and 0.41 A for alpha-carbon atoms and for all atoms, respectively. Twenty-six water molecules fall within 0.25 A of matching water molecules associated with the second protein molecule. The reactive Ser70 is on a turn of 3(10) helix at the N terminus of a longer alpha-helix (72-83). The penicillin-binding site near this helix contains at least seven water molecules. Upon penicillin entry, a water molecule in the oxyanion hole, hydrogen-bonded between the N terminus of helix (80-83) and beta-strand (230-238), would be displaced by the oxygen atom of the beta-lactam carbonyl group. An unexpelled molecule of water is proposed to be the catalytic water required for penicillin hydrolysis. The water is hydrogen-bonded to Glu166, a conserved residue in all beta-lactamases, and it lies 3 A from the alpha-face of a previously modeled penicillin. The position of the water-Glu166 pair is stabilized in the active site by a cis peptide bond at Pro167.

Amino Acid Sequence↗
PubMed1990

Beta-lactamase of Bacillus licheniformis 749/C at 2 A resolution.

Two crystal forms (A and B) of the 29,500 Da Class A beta-lactamase (penicillinase) from Bacillus licheniformis 749/C have been examined crystallographically. The structure of B-form crystals has been solved to 2 A resolution, the starting model for which was a 3.5 A structure obtained from A-form crystals. The beta-lactamase has an alpha + beta structure with 11 helices and 5 beta-strands seen also in a penicillin target DD-peptidase of Streptomyces R61. Atomic parameters of the two molecules in the asymmetric unit were refined by simulated annealing at 2.0 A resolution. The R factor is 0.208 for the 27,330 data greater than 3 sigma (F), with water molecules excluded from the model. The catalytic Ser-70 is at the N-terminus of a helix and is within hydrogen bonding distance of conserved Lys-73. Also interacting with the Lys-73 are Asn-132 and the conserved Glu-166, which is on a potentially flexible helix-containing loop. The structure suggests the binding of beta-lactam substrates is facilitated by interactions with Lys-234, Thr-235, and Ala-237 in a conserved beta-strand peptide, which is antiparallel to the beta-lactam's acylamido linkage; an exposed cavity near Asn-170 exists for acylamido substituents. The reactive double bond of clavulanate-type inhibitors may interact with Arg-244 on the fourth beta-strand. A very similar binding site architecture is seen in the DD-peptidase.

Bacillus↗
PubMed1990

Different modes of vancomycin and D-alanyl-D-alanine peptidase binding to cell wall peptide and a possible role for the vancomycin resistance protein.

A comparison was made of the binding modes of the bacterial cell wall precursor L-lysyl-D-alanyl-D-alanine to the glycopeptide antibiotic vancomycin and to the D-alanyl-D-alanine-cleaving peptidase of Streptomyces sp. strain R61, a model for cell wall-synthesizing enzymes whose X-ray three-dimensional structure is established. In each of the two pairings (vancomycin with peptide and DD-peptidase with peptide), polypeptide backbones were antiparallel, and the antibiotic or enzyme enveloped the peptide substrate from opposite sides. Hydrogen-bonding groups on the substrate which are involved with the DD-peptidase were shown to be different from the ones reported from nuclear magnetic resonance studies to be involved with vancomycin. Because of steric hindrance, the binding of either molecule to the substrate prevents the binding of the other molecule. Binding to the substrate by a D-alanyl-D-alanine-recognizing protein in a manner similar to that used by the DD-peptidase could explain recent observations of vancomycin resistance, in which a new membrane-associated protein has been detected.

Bacterial Proteins↗
PubMed1989

Crystallographic mapping of beta-lactams bound to a D-alanyl-D-alanine peptidase target enzyme.

X-ray crystallography has been used to examine the binding of three members of the beta-lactam family of antibiotics to the D-alanyl-D-alanine peptidase from Streptomyces R61, a target of penicillins. Cephalosporin C, the monobactam analog of penicillin G and (2,3)-alpha-methylene benzylpenicillin have been mapped at 2.3 A resolution in the form of acyl-enzyme complexes bound to serine 62. On the basis of the positions of these inhibitors, the binding of a tripeptide substrate for the enzyme, L-lysyl-D-alanyl-D-alanine, has been modeled in the active site. The binding of both inhibitors and substrate is facilitated by hydrogen-bonding interactions with a conserved beta-strand (297-303), which is antiparallel to the beta-lactam's acylamide linkage or the substrate's peptide bond. The active site is similar to that in beta-lactamases.

Anti-Bacterial Agents↗
PubMed1989

D-alanine-D-alanine ligase (ADP) from Salmonella typhimurium. Overproduction, purification, crystallization and preliminary X-ray analysis.

The ddlA gene from Salmonella typhimurium coding for D-alanine-D-alanine ligase (ADP-forming) has been subcloned behind the tac promotor in the plasmid pKK223-3, with expression in Escherichia coli JM105. The overexpression system yields 58 mg of active enzyme from 12 g of wet cell paste after 40-fold purification to homogeneity. 5,5'-Dithiobis-(2-nitrobenzoic acid) titrations indicate that all four cysteine residues exist as free thiols. Two crystal forms of the 39,300 Mr enzyme have been produced. A tetragonal form grows at 21 degrees C from 10 to 15% (w/v) polyethylene glycol 8000 in space group P4(1)2(1)2, with two molecules in the asymmetric unit; it has cell constants a = b = 83.8 A, c = 220.0 A, and diffracts to 2.9 A. A monoclinic form grows from 30% (w/v) ammonium sulfate in space group P2(1), with two molecules in the asymmetric unit; it has cell constants a = 60.4 A, b = 102.1 A, c = 64.3 A, beta = 115.7 degrees, and diffracts to 2.2 A resolution.

Cloning, Molecular↗
PubMed1989

Studying enzyme-beta-lactam interactions using X-ray diffraction.

The interaction of representative beta-lactam antibiotics with a bacterial enzyme target has been mapped in three dimensions using X-ray diffraction data to 2.25 A resolution. Examination of complexes of cephalosporin C, benzylmonobactam, and alpha-(2,3)-methylenepenicillin G with the D-alanyl-D-alanine transpeptidase-carboxypeptidase from Streptomyces R61 shows that the enzyme's reactive serine has acylated the beta-lactam ring of each inhibitor. The known half-lives of the three acyl complexes can be correlated with the distance of the drug's carboxylate (or sulfonate) group from complementary groups on the DD-peptidase.

Anti-Bacterial Agents↗
PubMed1987

Crystallization and preliminary X-ray and optical spectroscopic characterization of the photochemical reaction center from Rhodobacter sphaeroides strain 2.4.1.

The photochemical reaction center from Rhodobacter sphaeroides 2.4.1 has been crystallized. The crystals were obtained in a solution of beta-octylglucoside by the vapor diffusion technique using polyethylene glycol 4000 as the precipitant at 22 degrees C. The orthorhombic crystals (space group P2(1)2(1)2(1)) have cell constants a = 142.5 A, b = 136.1 A, c = 78.5 A, and diffract to 3.7 A. The crystals display pronounced linear dichroism in the carotenoid absorption spectral region.

Bacterial Proteins↗
PubMed1986

The association behaviour of beta-lactamases. Sedimentation equilibrium studies in ammonium sulphate solutions.

The beta-lactamases (EC 3.5.2.6) from TEM plasmid RP4, Bacillus licheniformis 749/C and Enterobacter cloacae P99 were studied in solution over a wide concentration range by equilibrium sedimentation. Though crystal symmetries indicate that all three enzymes are potentially dimeric in their crystal forms, in 50 mM-sodium cacodylate at pH 6.5 the enzymes show only a small tendency to associate, indicated by a weight-average Mr (Mw) at 3% (w/v) concentration about 9% greater than that of the monomer. Although the mode of association could not be determined, this extent of association corresponded to a dimerization constant of about 2 X 10(2) M-1. In 2.1 M-(NH4)2SO4 the B. licheniformis enzyme shows some association at concentrations over 1%, displaying an Mw value at 7% concentration about 60% more than the monomer. Under the same conditions Mw for the Entero. P99 enzyme is about 60% greater than the monomer near the solubility limit of about 2%. However, the Mw for the TEM enzyme is over twice that of the monomer at its solubility limit (3%) in 1.7 M-(NH4)2SO4. Fitting the sedimentation data of the TEM enzyme in 1.7 M-(NH4)2SO4 with a dimerization model and an indefinite-isodesmic-association model yielded equilibrium constants of 1.5 X 10(4) and 3.3 X 10(2) M-1 respectively, with the indefinite-isodesmic model giving the better fit. Fitting the data for the other two enzymes yielded values of 1.4 X 10(3) and 1.7 X 10(2) M-1 respectively for the Entero. P99 enzyme and 4.5 X 10(2) and 45 M-1 respectively for the B. licheniformis enzyme. It could not be determined which model was the better fit for these two enzymes. Since none of the beta-lactamases studied here showed strong evidence of the terminal aggregate being a dimer, we conclude that crystalline dimers, if they exist, will not be tightly associated or physiologically significant.

Ammonium Sulfate↗
PubMed1986

On the origin of bacterial resistance to penicillin: comparison of a beta-lactamase and a penicillin target.

Structural data are now available for comparing a penicillin target enzyme, the D-alanyl-D-alanine-peptidase from Streptomyces R61, with a penicillin-hydrolyzing enzyme, the beta-lactamase from Bacillus licheniformis 749/C. Although the two enzymes have distinct catalytic properties and lack relatedness in their overall amino acid sequences except near the active-site serine, the significant similarity found by x-ray crystallography in the spatial arrangement of the elements of secondary structure provides strong support for earlier hypotheses that beta-lactamases arose from penicillin-sensitive D-alanyl-D-alanine-peptidases involved in bacterial wall peptidoglycan metabolism.

Amino Acid Sequence↗
PubMed1985

2.8-A Structure of penicillin-sensitive D-alanyl carboxypeptidase-transpeptidase from Streptomyces R61 and complexes with beta-lactams.

The crystallographic structure of the penicillin-sensitive D-alanyl carboxypeptidase-transpeptidase from Streptomyces R61 has been solved to 2.8-A resolution. The 38,000-dalton serine peptidase has two regions of secondary structure, an alpha/beta cluster, and a region which contains five helical segments. The beta sheet is composed of five beta strands. The tertiary structure has no homology with the classic serine proteases or with the zinc carboxypeptidases. The binding at a common site of three types of beta-lactam (a penicillin, a cephalosporin, a monocyclic beta-lactam) and a desazacyclobutanone has been observed in Fourier difference maps. The binding site sequence is Val-Gly-Ser-Val-Thr-Lys. The beta-lactam ring lies near the enzyme's catalytic serine at position 37, and the C3 substituent of a cephalosporin falls near lysine 40.

Anti-Bacterial Agents↗
PubMed1985

Crystallization and preliminary X-ray data for the exocellular beta-lactamase of Bacillus licheniformis 749/C.

The exocellular beta-lactamase from Bacillus licheniformis 749/C has been crystallized from polyethylene glycol solution at pH 5.5. An X-ray examination of the monoclinic crystals shows the space group is P21, with unit cell dimensions a = 66.77 A, b = 93.77 A, c = 43.57 A and beta = 104.5 degrees. The asymmetric unit consists of two molecules of 28,500 Mr each. The crystals are suitable for structure analysis to at least 2 A resolution.

Bacillus↗
PubMed1979

Predicted secondary structures of four penicillin beta-lactamases and a comparison with two lysozymes.

We have predicted the secondary structures of four beta-lactamases (Bacillus cereus, Bacillus licheniformis, Staphylococcus aureus, and Escherichia coli R-TEM) by the statistical method of Chou & Fasman as well as by the information theory method of Garnier et al. The secondary structures of all four beta-lactamases are of the alpha/beta type (Levitt & Chothia's nomenclature), with helices at N- and C-termini. There are about eight short regions each of alpha-helical (30--50%) and beta-strand (10--20%) structure separated by about 20 reverse turns. The conformation of the Gram-positive and Gram-negative beta-lactamases are generally similar although a few differences are predicted between the S.aureus and E.coli structures. Surprisingly, the two bacilli structures differ significantly in three short regions. In all four enzymes the region near the catalytically-implicated tyrosine has similar secondary structure. The secondary structure of hen egg white lysozyme, a penicillin-binding enzyme, as well as T4 phage lysozyme, has similarities to the N-terminal half of the penicillin-destroying beta-lactamases.

Amino Acid Sequence↗