James R. Knox

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

Ultrahigh resolution structure of a class A β-lactamase: On the mechanism and specificity of the extended-spectrum SHV-2 enzyme

Bacterial beta-lactamases hydrolyze beta-lactam antibiotics such as penicillins and cephalosporins. The TEM-type class A beta-lactamase SHV-2 is a natural variant that exhibits activity against third-generation cephalosporins normally resistant to hydrolysis by class A enzymes. SHV-2 contains a single Gly238Ser change relative to the wild-type enzyme SHV-1. Crystallographic refinement of a model including hydrogen atoms gave R and R(free) of 12.4% and 15.0% for data to 0.91 A resolution. The hydrogen atom on the O(gamma) atom of the reactive Ser70 is clearly seen for the first time, bridging to the water molecule activated by Glu166. Though hydrogen atoms on the nearby Lys73 are not seen, this observation of the Ser70 hydrogen atom and the hydrogen bonding pattern around Lys73 indicate that Lys73 is protonated. These findings support a role for the Glu166-water couple, rather than Lys73, as the general base in the deprotonation of Ser70 in the acylation process of class A beta-lactamases. Overlay of SHV-2 with SHV-1 shows a significant 1-3 A displacement in the 238-242 beta-strand-turn segment, making the beta-lactam binding site more open to newer cephalosporins with large C7 substituents and thereby expanding the substrate spectrum of the variant enzyme. The OH group of the buried Ser238 side-chain hydrogen bonds to the main-chain CO of Asn170 on the Omega loop, that is unaltered in position relative to SHV-1. This structural role for Ser238 in protein-protein binding makes less likely its hydrogen bonding to oximino cephalosporins such as cefotaxime or ceftazidime.

Comparison of β-lactamases of classes A and D : 1.5-Å crystallographic structure of the class D OXA-1 oxacillinase

The crystallographic structure of the Escherichia coli OXA‐1 β‐lactamase has been established at 1.5‐Å resolution and refined to R = 0.18. The 28.2‐kD oxacillinase is a class D serine β‐lactamase that is especially active against the penicillin‐type β‐lactams oxacillin and cloxacillin. In contrast to the structures of OXA‐2, OXA‐10, and OXA‐13 belonging to other subclasses, the OXA‐1 molecule is monomeric rather than dimeric and represents the subclass characterized by an enlarged Ω loop near the β‐lactam binding site. The 6‐residue hydrophilic insertion in this loop cannot interact directly with substrates and, instead, projects into solvent. In this structure at pH 7.5, carboxylation of the conserved Lys 70 in the catalytic site is observed. One oxygen atom of the carboxylate group is hydrogen bonded to Ser 120 and Trp 160. The other oxygen atom is more exposed and hydrogen bonded to the Oγ of the reactive Ser 67. In the overlay of the class D and class A binding sites, the carboxylate group is displaced ca. 2.6 Å from the carboxylate group of Glu 166 of class A enzymes. However, each group is equidistant from the site of the water molecule expected to function in hydrolysis, and which could be activated by the carboxylate group of Lys 70. In this ligand‐free OXA‐1 structure, no water molecule is seen in this site, so the water molecule must enter after formation of the acyl‐Ser 67 intermediate.

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.

Hydrolysis of Third-generation Cephalosporins by Class C β-Lactamases STRUCTURES OF A TRANSITION STATE ANALOG OF CEFOTAXIME IN WILD-TYPE AND EXTENDED SPECTRUM ENZYMES

Bacterial resistance to the third-generation cephalosporins is an issue of great concern in current antibiotic therapeutics. An important source of this resistance is from production of extended-spectrum (ES) β-lactamases by bacteria. The Enterobacter cloacae GC1 enzyme is an example of a class C ES β-lactamase. Unlike wild-type (WT) forms, such as the E. cloacae P99 and Citrobacter freundii enzymes, the ES GC1 β-lactamase is able to rapidly hydrolyze third-generation cephalosporins such as cefotaxime and ceftazidime. To understand the basis for this ES activity, m-nitrophenyl 2-(2-aminothiazol-4-yl)-2-[(Z)-methoxyimino]acetylaminomethyl phosphonate has been synthesized and characterized. This phosphonate was designed to generate a transition state analog for turnover of cefotaxime. The crystal structures of complexes of the phosphonate with both ES GC1 and WT C. freundii GN346 β-lactamases have been determined to high resolution (1.4–1.5 Å). The serine-bound analog of the tetrahedral transition state for deacylation exhibits a very different binding geometry in each enzyme. In the WT β-lactamase the cefotaxime-like side chain is crowded against the Ω loop and must protrude from the binding site with its methyloxime branch exposed. In the ES enzyme, a mutated Ω loop adopts an alternate conformation allowing the side chain to be much more buried. During the binding and turnover of the cefotaxime substrate by this ES enzyme, it is proposed that ligand-protein contacts and intra-ligand contacts are considerably relieved relative to WT, facilitating positioning and activation of the hydrolytic water molecule. The ES β-lactamase is thus able to efficiently inactivate third-generation cephalosporins.

Enzymes of vancomycin resistance: the structure of D-alanine-D-lactate ligase of naturally resistant Leuconostoc mesenteroides.

BACKGROUND The bacterial cell wall and the enzymes that synthesize it are targets of glycopeptide antibiotics (vancomycins and teicoplanins) and beta-lactams (penicillins and cephalosporins). Biosynthesis of cell wall peptidoglycan requires a crosslinking of peptidyl moieties on adjacent glycan strands. The D-alanine-D-alanine transpeptidase, which catalyzes this crosslinking, is the target of beta-lactam antibiotics. Glycopeptides, in contrast, do not inhibit an enzyme, but bind directly to D-alanine-D-alanine and prevent subsequent crosslinking by the transpeptidase. Clinical resistance to vancomycin in enterococcal pathogens has been traced to altered ligases producing D-alanine-D-lactate rather than D-alanine-D-alanine. RESULTS The structure of a D-alanine-D-lactate ligase has been determined by multiple anomalous dispersion (MAD) phasing to 2.4 A resolution. Co-crystallization of the Leuconostoc mesenteroides LmDdl2 ligase with ATP and a di-D-methylphosphinate produced ADP and a phosphinophosphate analog of the reaction intermediate of cell wall peptidoglycan biosynthesis. Comparison of this D-alanine-D-lactate ligase with the known structure of DdlB D-alanine-D-alanine ligase, a wild-type enzyme that does not provide vancomycin resistance, reveals alterations in the size and hydrophobicity of the site for D-lactate binding (subsite 2). A decrease was noted in the ability of the ligase to hydrogen bond a substrate molecule entering subsite 2. CONCLUSIONS Structural differences at subsite 2 of the D-alanine-D-lactate ligase help explain a substrate specificity shift (D-alanine to D-lactate) leading to remodeled cell wall peptidoglycan and vancomycin resistance in Gram-positive pathogens.

Order parameter measurements in polypeptide liquid crystals

An analysis of x‐ray scattering from magnetically oriented liquid crystalline solutions of poly‐γ‐benzyl‐L‐glutamate (PBLG) in dioxane yields a value for the order parameter S?0.75. At the isotropic–liquid crystal phase transition, the critical order parameter Sc?0.5 appears to agree with predictions of mean‐field theory. Away from the transition, S is independent of PBLG concentration and PBLG axial ratio but does decrease when the helix denaturing agent trifluoroacetic acid is added to the liquid crystal. The magnitude of and changes in S are discussed in terms of the inherent flexibility of helical synthetic polypeptides.

β-Lactamase mutations far from the active site influence inhibitor binding

Analysis of the three dimensional structure of the class A beta-lactamases shows that Arg-244, a spatially conserved residue important for inactivation by clavulanic acid, is held in place by a hydrogen (H) bond from the residue at 276. An Asn276-Gly mutant of OHIO-1, an SHV family class A enzyme, was constructed to investigate the importance of that interaction. Compared to a strain expressing the wild type enzyme, OHIO-1, the MIC of the Asn276-Gly mutant strain was more resistant to clavulanate (0.25 vs. 2.0 micrograms/ml) in the presence of ampicillin (16 micrograms/ml) but was as susceptible to sulbactam or tazobactam plus ampicillin as the OHIO-1 bearing strain. No difference in MICs was observed when other beta-lactams were tested. Consistent with the susceptibility test results, the apparent Ki of clavulanate for the Asn276-Gly enzyme (4.5 microM) was 10-fold greater than OHIO-1 (0.4 microM). For sulbactam and tazobactam the apparent Ki decreased for Asn276-Gly enzyme (1.0 and 0.1 micrograms/ml, respectively) compared to the wild-type parent (17 and 0.7 micrograms/ml, respectively). Comparing the Asn276-Gly heta-lactamase with OHIO-1, the Vmax for most substrates except cephaloridine did not change substantially. There was a 2-15 fold decreased affinity (Km) and catalytic efficiency (Vmax/Km) for beta-lactam substrates. These data support the observation and emphasize the role for this H bonding residue in orienting Arg-244 towards the active site.