Judith A. Kelly

Comparison of the sequences of class A beta-lactamases and of the secondary structure elements of penicillin-recognizing proteins.

The sequences of class A beta-lactamases were compared. Four main groups of enzymes were distinguished: those from the gram-negative organisms and bacilli and two distinct groups of Streptomyces spp. The Staphylococcus aureus PC1 enzyme, although somewhat closer to the enzyme from the Bacillus group, did not belong to any of the groups of beta-lactamases. The similarities between the secondary structure elements of these enzymes and those of the class C beta-lactamases and of the Streptomyces sp. strain R61 DD-peptidase were also analyzed and tentatively extended to the class D beta-lactamases. A unified nomenclature of secondary structure elements is proposed for all the penicillin-recognizing enzymes.

Crystallographic mapping of β-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-lactams acylamide linkage or the substrates peptide bond. The active site is similar to that in beta-lactamases.

Structures of two kinetic intermediates reveal species specificity of penicillin-binding proteins.

Penicillin-binding proteins (PBPs), the target enzymes of beta-lactam antibiotics such as penicillins and cephalosporins, catalyze the final peptidoglycan cross-linking step of bacterial cell-wall biosynthesis. beta-Lactams inhibit this reaction because they mimic the D-alanyl-D-alanine peptide precursors of cell-wall structure. Prior crystallographic studies have described the site of beta-lactam binding and inhibition, but they have failed to show the binding of D-Ala-D-Ala substrates. We present here the first high-resolution crystallographic structures of a PBP, D-Ala-D-Ala-peptidase of Streptomyces sp. strain R61, non-covalently complexed with a highly specific fragment (glycyl-L-alpha-amino-epsilon-pimelyl-D-Ala-D-Ala) of the cell-wall precursor in both enzyme-substrate and enzyme-product forms. The 1.9A resolution structure of the enzyme-substrate Henri-Michaelis complex was achieved by using inactivated enzyme, which was formed by cross-linking two catalytically important residues Tyr159 and Lys65. The second structure at 1.25A resolution of the uncross-linked, active form of the DD-peptidase shows the non-covalent binding of the two products of the carboxypeptidase reaction. The well-defined substrate-binding site in the two crystallographic structures shows a subsite that is complementary to a portion of the natural cell-wall substrate that varies among bacterial species. In addition, the structures show the displacement of 11 water molecules from the active site, the location of residues responsible for substrate binding, and clearly demonstrate the necessity of Lys65 and or Tyr159 for the acylation step with the donor peptide. Comparison of the complexed structures described here with the structures of other known PBPs suggests the design of species-targeted antibiotics as a counter-strategy towards beta-lactamase-elicited bacterial resistance.

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.

X-RAY STUDIES OF ENZYMES THAT INTERACT WITH PENICILLINS

Abstract. The technique of X-ray diffraction has been successfully applied to enzymes associated with peptidoglycan biosynthesis. The technique has taught us a great deal about the structures and catalytic mechanisms of penicillin-binding proteins and β-lactamases. An insight into the structural basis for antibiotic resistance is given.

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.

Studying enzyme-β-lactam interactions using X-ray diffraction*

The interaction of representative β-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 α-(2,3)-methylenepenicillin G with the D-alanyl-D-alanine transpeptidase-carboxypeptidase from Streptomyces R61 shows that the enzymes reactive serine has acylated the β-lactam ring of each inhibitor. The known half-lives of the three acyl complexes can be correlated with the distance of the drugs carboxylate (or sulfonate) group from complementary groups on the DD-peptidase.