Background:Avibactam is a ␤-lactamase inhibitor with a broad spectrum of activity. Results: Kinetic parameters of inhibition as well as acyl enzyme stability are reported against six clinically relevant enzymes. Conclusion: Inhibition efficiency is highest against class A, then class C, and then class D. Significance: These base-line inhibition values across enzyme classes provide the foundation for future structural and mechanistic enzymology experiments.

dAlthough ␤-lactams have been the most effective class of antibacterial agents used in clinical practice for the past half century, their effectiveness on Gram-negative bacteria has been eroded due to the emergence and spread of ␤-lactamase enzymes that are not affected by currently marketed ␤-lactam/␤-lactamase inhibitor combinations. Avibactam is a novel, covalent, non-␤-lactam ␤-lactamase inhibitor presently in clinical development in combination with either ceftaroline or ceftazidime. In vitro studies show that avibactam may restore the broad-spectrum activity of cephalosporins against class A, class C, and some class D ␤-lactamases. Here we describe the structures of two clinically important ␤-lactamase enzymes bound to avibactam, the class A CTX-M-15 extended-spectrum ␤-lactamase and the class C Pseudomonas aeruginosa AmpC ␤-lactamase, which together provide insight into the binding modes for the respective enzyme classes. The structures reveal similar binding modes in both enzymes and thus provide a rationale for the broad-spectrum inhibitory activity of avibactam. Identification of the key residues surrounding the binding pocket allows for a better understanding of the potency of this scaffold. Finally, avibactam has recently been shown to be a reversible inhibitor, and the structures provide insights into the mechanism of avibactam recyclization. Analysis of the ultra-high-resolution CTX-M-15 structure suggests how the deacylation mechanism favors recyclization over hydrolysis.

Enzymes provide enormous rate enhancements, unmatched by any other type of catalyst. The stabilization of high-energy states along the reaction coordinate is the crux of the catalytic power of enzymes. We report the atomic-resolution structure of a high-energy reaction intermediate stabilized in the active site of an enzyme. Crystallization of phosphorylated beta-phosphoglucomutase in the presence of the Mg(II) cofactor and either of the substrates glucose 1-phosphate or glucose 6-phosphate produced crystals of the enzyme-Mg(II)-glucose 1,6-(bis)phosphate complex, which diffracted x-rays to 1.2 and 1.4 angstroms, respectively. The structure reveals a stabilized pentacovalent phosphorane formed in the phosphoryl transfer from the C(1)O of glucose 1,6-(bis)phosphate to the nucleophilic Asp8 carboxylate.

Multidrug-resistant (MDR) bacterial infections are a serious threat to public health. Among the most alarming resistance trends is the rapid rise in the number and diversity of β-lactamases, enzymes that inactivate β-lactams, a class of antibiotics that has been a therapeutic mainstay for decades. Although several new β-lactamase inhibitors have been approved or are in clinical trials, their spectra of activity do not address MDR pathogens such as Acinetobacter baumannii. This report describes the rational design and characterization of expanded-spectrum serine β-lactamase inhibitors that potently inhibit clinically relevant class A, C and D β-lactamases and penicillin-binding proteins, resulting in intrinsic antibacterial activity against Enterobacteriaceae and restoration of β-lactam activity in a broad range of MDR Gram-negative pathogens. One of the most promising combinations is sulbactam-ETX2514, whose potent antibacterial activity, in vivo efficacy against MDR A. baumannii infections and promising preclinical safety demonstrate its potential to address this significant unmet medical need.

Creatine kinase (CK) catalyzes the reversible conversion of creatine and ATP to phosphocreatine and ADP, thereby helping maintain energy homeostasis in the cell. Here we report the first X-ray structure of CK bound to a transition-state analogue complex (CK-TSAC). Cocrystallization of the enzyme from Torpedo californica (TcCK) with ADP-Mg(2+), nitrate, and creatine yielded a homodimer, one monomer of which was liganded to a TSAC complex while the second monomer was bound to ADP-Mg(2+) alone. The structures of both monomers were determined to 2.1 A resolution. The creatine is located with the guanidino nitrogen cis to the methyl group positioned to perform in-line attack at the gamma-phosphate of ATP-Mg(2+), while the ADP-Mg(2+) is in a conformation similar to that found in the TSAC-bound structure of the homologue arginine kinase (AK). Three ligands to Mg(2+) are contributed by ADP and nitrate and three by ordered water molecules. The most striking difference between the substrate-bound and TSAC-bound structures is the movement of two loops, comprising residues 60-70 and residues 323-332. In the TSAC-bound structure, both loops move into the active site, resulting in the positioning of two hydrophobic residues (one from each loop), Ile69 and Val325, near the methyl group of creatine. This apparently provides a specificity pocket for optimal creatine binding as this interaction is missing in the AK structure. In addition, the active site of the transition-state analogue complex is completely occluded from solvent, unlike the ADP-Mg(2+)-bound monomer and the unliganded structures reported previously.

Phosphoglucomutases catalyze the interconversion of D-glucose 1-phosphate and D-glucose 6-phosphate, a reaction central to energy metabolism in all cells and to the synthesis of cell wall polysaccharides in bacterial cells. Two classes of phosphoglucomutases (R-PGM and -PGM) are distinguished on the basis of their specificity for R-and -glucose-1-phosphate. -PGM is a member of the haloacid dehalogenase (HAD) superfamily, which includes the sarcoplasmic Ca 2+ -ATPase, phosphomannomutase, and phosphoserine phosphatase. -PGM is unusual among family members in that the common phosphoenzyme intermediate exists as a stable ground-state complex in this enzyme. Herein we report, for the first time, the three-dimensional structure of a -PGM and the first view of the true phosphoenzyme intermediate in the HAD superfamily. The crystal structure of the Mg(II) complex of phosphorylated -phosphoglucomutase ( -PGM) from Lactococcus lactis has been determined to 2.3 Å resolution by multiwavelength anomalous diffraction (MAD) phasing on selenomethionine, and refined to an R cryst ) 0.24 and R free ) 0.28. The active site of -PGM is located between the core and the cap domain and is freely solvent accessible. The residues within a 6 Å radius of the phosphorylated Asp8 include Asp10, Thr16, Ser114, Lys145, Glu169, and Asp170. The cofactor Mg 2+ is liganded with octahedral coordination geometry by the carboxylate side chains of Asp8, Glu169, Asp170, and the backbone carbonyl oxygen of Asp10 along with one oxygen from the Asp8-phosphoryl group and one water ligand. The phosphate group of the phosphoaspartyl residue, Asp8, interacts with the side chains of Ser114 and Lys145. The absence of a base residue near the aspartyl phosphate group accounts for the persistence of the phosphorylated enzyme under physiological conditions. Substrate docking shows that glucose-6-P can bind to the active site of phosphorylated -PGM in such a way as to position the C(1)OH near the phosphoryl group of the phosphorylated Asp8 and the C(6) phosphoryl group near the carboxylate group of Asp10. This result suggests a novel two-base mechanism for phosphoryl group transfer in a phosphorylated sugar.In this paper, we report the three-dimensional structure of the -phosphoglucomutase from Lactococcus lactis. Both R-and -phosphoglucomutases catalyze the interconversion of D-glucose-1-phosphate (G1P) and glucose-6-phosphate (G6P). This reaction is central to energy metabolism in all cells and is essential to the synthesis of cell wall polysaccharides in bacterial cells (1, 2). The R-phosphoglucomutase (R-PGM) acts on the R-C(1) anomer of G1P, while the -phosphoglucomutase ( -PGM) catalyzes the reaction of the -C(1) anomer. Both mutases employ Mg 2+ and -or R-glucose 1,6-diphosphate (G1,6-diP) as cofactors (1, 2). In addition, both mutases are monomeric proteins. The R-PGM (65 kDa) is, however, approximately twice the size of the -PGM (25 kDa) (3). The X-ray structure of R-PGM from rat reveals a 4-domain R-/ -protein. All four domains contribute residue...

bAvibactam is a novel non-␤-lactam ␤-lactamase inhibitor that inhibits a wide range of ␤-lactamases. These include class A, class C, and some class D enzymes, which erode the activity of ␤-lactam drugs in multidrug-resistant pathogens like Pseudomonas aeruginosa and Enterobacteriaceae spp. Avibactam is currently in clinical development in combination with the ␤-lactam antibiotics ceftazidime, ceftaroline fosamil, and aztreonam. Avibactam has the potential to be the first ␤-lactamase inhibitor that might provide activity against class C-mediated resistance, which represents a growing concern in both hospital-and community-acquired infections. Avibactam has an unusual mechanism of action: it is a covalent inhibitor that acts via ring opening, but in contrast to other currently used ␤-lactamase inhibitors, this reaction is reversible. Here, we present a high-resolution structure of avibactam bound to a class C ␤-lactamase, AmpC, from P. aeruginosa that provided insight into the mechanism of both acylation and recyclization in this enzyme class and highlighted the differences observed between class A and class C inhibition. Furthermore, variants resistant to avibactam that identified the residues important for inhibition were isolated. Finally, the structural information was used to predict effective inhibition by sequence analysis and functional studies of class C ␤-lactamases from a large and diverse set of contemporary clinical isolates (P. aeruginosa and several Enterobacteriaceae spp.) obtained from recent infections to understand any preexisting variability in the binding pocket that might affect inhibition by avibactam.

The Class D (or OXA-type) β-lactamases have expanded to be the most diverse group of serine β-lactamases with a highly heterogeneous β-lactam hydrolysis profile and are typically resistant to marketed β-lactamase inhibitors. Class D enzymes are increasingly found in multidrug resistant (MDR) Acinetobacter baumannii, Pseudomonas aeruginosa, and various species of the Enterobacteriaceae and are posing a serious threat to the clinical utility of β-lactams including the carbapenems, which are typically reserved as the drugs of last resort. Avibactam, a novel non-β-lactam β-lactamase inhibitor, not only inhibits all class A and class C β-lactamases but also has the promise of inhibition of certain OXA enzymes, thus extending the antibacterial activity of the β-lactam used in combination to the organisms that produce these enzymes. X-ray structures of OXA-24 and OXA-48 in complex with avibactam revealed the binding mode of this inhibitor in this diverse class of enzymes and provides a rationale for selective inhibition of OXA-48 members. Additionally, various subunits of the OXA-48 structure in the asymmetric unit provide snapshots of different states of the inhibited enzyme. Overall, these data provide the first structural evidence of the exceptionally slow reversibility observed with avibactam in class D β-lactamases. Mechanisms for acylation and deacylation of avibactam by class D enzymes are proposed, and the likely extent of inhibition of class D β-lactamases by avibactam is discussed.