Paul C. Moews

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.

Molecular evolution of bacterial β-lactam resistance

Abstract Background: Two groups of penicillin-destroying enzymes, the class A and class C β-lactamases, may have evolved from bacterial transpeptidases that transfer x-d-Ala-d-Ala peptides to the growing peptidoglycan during cell wall synthesis. Both the transpeptidases and the β-lactamases are acylated by β-lactam antibiotics such as penicillin, which mimic the peptide, but breakdown and removal of the antibiotic is much faster in the β-lactamases, which lack the ability to process d-Ala-d-Ala peptides. Stereochemical factors driving this evolution in specificity are examined. Results: We have compared the crystal structures of two classes of β-lactamases and a β-lactam-sensitive d-alanyl-d-alanine-carboxypeptidase/transpeptidase (DD-peptidase). The class C β-lactamase is more similar to the DD-peptidase than to another β-lactamase of class A. Conclusions: The two classes of β-lactamases appear to have developed from an ancestral protein along separate evolutionary paths. Structural differentiation of the β-lactamases from the DD-peptidases appears to follow differences in substrate shapes. The structure of the class A β-lactamase has been further optimized to exclude d-alanyl peptides and process penicillin substrates with near catalytic perfection. Keywords: drug resistance, enzymology, penicillin antibiotics, protein ancestry Received: 7 October 1996

5.5 Å crystallographic structure of penicillin β-lactamase and radius of gyration in solution☆

Abstract An electron density map of crystalline R-TEM Escherichia coli β-lactamase (penicillinase) has been calculated from X-ray diffraction data at 5.5 A resolution with protein phases based on Friedel mates from a high-quality samarium derivative. The mean figure of merit for 854 independent reflections is 0.75. The monomeric molecule is slightly ellipsoidal and contains one and possibly two regions of α-helix which are 25 A long. The Crystallographic search for the substrate binding site has so far been inconclusive. The radius of gyration of the enzyme in solution at pH 7 is 17.1 ± 1.0 A from small-angle X-ray scattering measurements. This compares with 18.6 a calculated from the low-resolution electron density map of the molecule in the crystal.

Crystallographic data for the β-lactamase from Enterobacter cloacae P99

The β-lactamase from Enterobacter cloacae P99 has been crystallized from polyethylene glycol solution at pH 7. X-ray examination of the orthorhombic crystals shows the space group is P 2 1 2 1 2 with unit cell dimensions a =77·4 , b =69· , and c =63·6 . There is one molecule of molecular weight 39,000 in the asymmetric unit.

Crystallographic data for a penicillin receptor: Exocellular dd-carboxypeptidase-transpeptidase from Streptomyces R61☆

Abstract A pencillin-sensitive enzyme, the exocellular dd -carboxypeptidase-transpeptidase from Streptomyces R61, has been crystallized from polyethylene glycol (Mr = 6000 to 7500) solution at pH 7·6. X-ray examination of the orthorhombic crystals shows the space group is P212121, with unit cell dimensions a = 51·1 A , b = 67·4 A , and c = 102·9 A . With four molecules of molecular weight 38,000, the A 3 / dalton ratio for the cell is 2·33. The crystals are stable to irradiation for 75 hours and are suitable for structure analysis to at least 2·4 A resolution. The radius of gyration of the molecule in solution at pH 6.8 is 20.8 A.