Christopher T. Lohans

Structural and stereoelectronic insights into oxygenase-catalyzed formation of ethylene from 2-oxoglutarate.

Significance The plant-signaling molecule ethylene is biosynthesized from 1-aminocyclopropane-1-carboxylic acid (ACC), as catalyzed by ACC oxidase, which is homologous to the 2-oxoglutarate (2OG) oxygenases, but which does not use a 2OG cosubstrate. Bacteria produce ethylene in a highly unusual reaction that involves oxidative 2OG fragmentation. Biophysical studies on a Pseudomonas ethylene-forming enzyme (EFE) reveal how structural and stereoelectronic factors enable the EFE to bias reaction away from normal 2OG oxygenase catalysis involving two-electron substrate oxidation concomitant with succinate formation, toward the arginine-dependent four-electron oxidation of 2OG to give ethylene. The results imply that negative catalysis, with respect to ethylene formation, has operated during the evolution of 2OG oxygenases and will be useful in protein engineering aimed at optimizing ethylene production. Ethylene is important in industry and biological signaling. In plants, ethylene is produced by oxidation of 1-aminocyclopropane-1-carboxylic acid, as catalyzed by 1-aminocyclopropane-1-carboxylic acid oxidase. Bacteria catalyze ethylene production, but via the four-electron oxidation of 2-oxoglutarate to give ethylene in an arginine-dependent reaction. Crystallographic and biochemical studies on the Pseudomonas syringae ethylene-forming enzyme reveal a branched mechanism. In one branch, an apparently typical 2-oxoglutarate oxygenase reaction to give succinate, carbon dioxide, and sometimes pyrroline-5-carboxylate occurs. Alternatively, Grob-type oxidative fragmentation of a 2-oxoglutarate–derived intermediate occurs to give ethylene and carbon dioxide. Crystallographic and quantum chemical studies reveal that fragmentation to give ethylene is promoted by binding of l-arginine in a nonoxidized conformation and of 2-oxoglutarate in an unprecedented high-energy conformation that favors ethylene, relative to succinate formation.

Cyclic Boronates Inhibit All Classes of β-Lactamases

ABSTRACT β-Lactamase-mediated resistance is a growing threat to the continued use of β-lactam antibiotics. The use of the β-lactam-based serine-β-lactamase (SBL) inhibitors clavulanic acid, sulbactam, and tazobactam and, more recently, the non-β-lactam inhibitor avibactam has extended the utility of β-lactams against bacterial infections demonstrating resistance via these enzymes. These molecules are, however, ineffective against the metallo-β-lactamases (MBLs), which catalyze their hydrolysis. To date, there are no clinically available metallo-β-lactamase inhibitors. Coproduction of MBLs and SBLs in resistant infections is thus of major clinical concern. The development of “dual-action” inhibitors, targeting both SBLs and MBLs, is of interest, but this is considered difficult to achieve due to the structural and mechanistic differences between the two enzyme classes. We recently reported evidence that cyclic boronates can inhibit both serine- and metallo-β-lactamases. Here we report that cyclic boronates are able to inhibit all four classes of β-lactamase, including the class A extended spectrum β-lactamase CTX-M-15, the class C enzyme AmpC from Pseudomonas aeruginosa, and class D OXA enzymes with carbapenem-hydrolyzing capabilities. We demonstrate that cyclic boronates can potentiate the use of β-lactams against Gram-negative clinical isolates expressing a variety of β-lactamases. Comparison of a crystal structure of a CTX-M-15:cyclic boronate complex with structures of cyclic boronates complexed with other β-lactamases reveals remarkable conservation of the small-molecule binding mode, supporting our proposal that these molecules work by mimicking the common tetrahedral anionic intermediate present in both serine- and metallo-β-lactamase catalysis.

Structural/mechanistic insights into the efficacy of nonclassical β-lactamase inhibitors against extensively drug resistant Stenotrophomonas maltophilia clinical isolates

Clavulanic acid and avibactam are clinically deployed serine β‐lactamase inhibitors, important as a defence against antibacterial resistance. Bicyclic boronates are recently discovered inhibitors of serine and some metallo β‐lactamases. Here, we show that avibactam and a bicyclic boronate inhibit L2 (serine β‐lactamase) but not L1 (metallo β‐lactamase) from the extensively drug resistant human pathogen Stenotrophomonas maltophilia. X‐ray crystallography revealed that both inhibitors bind L2 by covalent attachment to the nucleophilic serine. Both inhibitors reverse ceftazidime resistance in S. maltophilia because, unlike clavulanic acid, they do not induce L1 production. Ceftazidime/inhibitor resistant mutants hyperproduce L1, but retain aztreonam/inhibitor susceptibility because aztreonam is not an L1 substrate. Importantly, avibactam, but not the bicyclic boronate is deactivated by L1 at a low rate; the utility of avibactam might be compromised by mutations that increase this deactivation rate. These data rationalize the observed clinical efficacy of ceftazidime/avibactam plus aztreonam as combination therapy for S. maltophilia infections and confirm that aztreonam‐like β‐lactams plus nonclassical β‐lactamase inhibitors, particularly avibactam‐like and bicyclic boronate compounds, have potential for treating infections caused by this most intractable of drug resistant pathogens.

New Delhi metallo-β-lactamase-1 catalyses avibactam and aztreonam hydrolysis

Metallo-lactamases (MBLs) threaten the clinical utility of -lactam antibiotics by hydrolyzing penicillins, cephalosporins, and carbapenems. Moreover, they can also hydrolyze all clinically used inhibitors (e.g., clavulanic acid, sulbactam, and tazobactam) that protect -lactam antibiotics from the activity of multidrug-resistant bacteria (1). Even the diazabicyclooctane (DBO)-based serine-lactamase (SBL) inhibitor avibactam, which was recently approved by the FDA, is hydrolyzed slowly by some MBLs (2). The combination of avibactam and the monobactam antibiotic aztreonam has recently passed phase II clinical trials for the treatment of infections by multidrugresistant Gram-negative bacteria producing MBLs (3). While SBL-mediated resistance to aztreonam has long been known via the evolution of SBLs (4), MBLs are not thought to hydrolyze aztreonam (5–7). Due to structural similarities between avibactam and aztreonam (Fig. 1A), particularly with respect to the sulfonate/sulfate substituent on the -lactam/urea nitrogen, we were interested in examining the interaction between more recently discovered MBLs and aztreonam and the potential for new clinically relevant MBLs with monobactam hydrolyzing activity. We tested the hydrolysis of aztreonam by recombinant enzymes covering all three subclasses of MBLs (i.e., B1, B2, and B3). Following overnight incubation of a 1:10 ratio of MBL and aztreonam, the extent of hydrolysis was determined by nuclear magnetic resonance (NMR) spectroscopy. While Verona integron-encoded MBL-1 (VIM-1) (subclass B1), VIM-4 (B1), CphA (B2), and L1 (B3) did not hydrolyze aztreonam (within our limits of detection), the model MBL BcII (B1) showed partial hydrolysis, and New Delhi MBL-1 (NDM-1) (B1) fully hydrolyzed aztreonam under our assay conditions (Fig. 1B). The BcII data are in broad agreement with the previously observed “nonproductive” binding of aztreonam to BcII (8). Interestingly, no interaction between aztreonam and NDM-1 was observed by 19F-NMR analysis (9), suggesting that the binding interaction (e.g., Km) is quite weak. Therefore, more detailed kinetic analyses were performed. The hydrolysis of aztreonam by NDM-1 was monitored over a shorter time scale (Fig. 1C), yielding a specific activity of 3.7 0.4 nmol min 1 mg 1 using 10 M NDM-1 and 1 mM aztreonam. The dependence of aztreonam hydrolysis on NDM-1 activity was confirmed by inhibition in the presence of EDTA and D-captopril, both inhibitors of MBLs (Fig. 1C). The hydrolysis of avibactam by NDM-1 was also shown by NMR analysis, which indicated that avibactam is hydrolyzed more quickly than aztreonam (Fig. 1C). The kinetics of avibactam and aztreonam hydrolysis by NDM-1 were further investigated by UV-visible (UV-Vis) spectroscopy and NMR spectroscopy (Fig. 1D). Due to poor substrate turnover and the limitations associated with these detection methods, a full kinetic characterization was not possible; while the values obtained are expected to be imprecise, they may serve as estimates of substrate affinity and turnover. Accepted manuscript posted online 2 October 2017 Citation Lohans CT, Brem J, Schofield CJ. 2017. New Delhi metallo-β-lactamase 1 catalyzes avibactam and aztreonam hydrolysis. Antimicrob Agents Chemother 61:e01224-17. https://doi.org/10.1128/AAC.01224-17. Copyright

A New Mechanism for β‐Lactamases: Class D Enzymes Degrade 1β‐Methyl Carbapenems through Lactone Formation

Abstract β‐Lactamases threaten the clinical use of carbapenems, which are considered antibiotics of last resort. The classical mechanism of serine carbapenemase catalysis proceeds through hydrolysis of an acyl‐enzyme intermediate. We show that class D β‐lactamases also degrade clinically used 1β‐methyl‐substituted carbapenems through the unprecedented formation of a carbapenem‐derived β‐lactone. β‐Lactone formation results from nucleophilic attack of the carbapenem hydroxyethyl side chain on the ester carbonyl of the acyl‐enzyme intermediate. The carbapenem‐derived lactone products inhibit both serine β‐lactamases (particularly class D) and metallo‐β‐lactamases. These results define a new mechanism for the class D carbapenemases, in which a hydrolytic water molecule is not required.