Restricted Rotational Flexibility of the C5α-Methyl-Substituted Carbapenem NA-1-157 Leads to Potent Inhibition of the GES-5 Carbapenemase.

Carbapenem antibiotics are used as a last-resort treatment for infections caused by multidrug-resistant bacteria. The wide spread of carbapenemases in Gram-negative bacteria has severely compromised the utility of these drugs and represents a serious public health threat. To combat carbapenemase-mediated resistance, new antimicrobials and inhibitors of these enzymes are urgently needed. Here, we describe the interaction of the atypically C5α-methyl-substituted carbapenem, NA-1-157, with the GES-5 carbapenemase. MICs of this compound against Escherichia coli, Klebsiella pneumoniae, and Acinetobacter baumannii producing the enzyme were reduced 4-16-fold when compared to MICs of the commercial carbapenems, reaching clinically sensitive breakpoints. When NA-1-157 was combined with meropenem, a strong synergistic effect was observed. Kinetic and ESI-LC/MS studies demonstrated that NA-1-157 is a potent inhibitor of GES-5, with a high inactivation efficiency of (2.9 ± 0.9) × 105 M-1 s-1. Acylation of GES-5 by NA-1-157 was biphasic, with the fast phase completing within seconds, and the slow phase taking several hours and likely proceeding through a reversible tetrahedral intermediate. Deacylation was extremely slow (k3 = (2.4 ± 0.3) × 10-7 s-1), resulting in a residence time of 48 ± 6 days. MD simulation of the GES-5-meropenem and GES-5-NA-1-157 acyl-enzyme complexes revealed that the C5α-methyl group in NA-1-157 sterically restricts rotation of the 6α-hydroxyethyl group preventing ingress of the deacylating water into the vicinity of the scissile bond of the acyl-enzyme intermediate. These data demonstrate that NA-1-157 is a potent irreversible inhibitor of the GES-5 carbapenemase.

The C5α-Methyl-Substituted Carbapenem NA-1-157 Exhibits Potent Activity against Klebsiella spp. Isolates Producing OXA-48-Type Carbapenemases.

The wide spread of carbapenem-hydrolyzing ß-lactamases in Gram-negative bacteria has diminished the utility of the last-resort carbapenem antibiotics, significantly narrowing the available therapeutic options. In the Enterobacteriaceae family, which includes many important clinical pathogens such as Klebsiella pneumoniae and Escherichia coli, production of class D ß-lactamases from the OXA-48-type family constitutes the major mechanism of resistance to carbapenems. To address the public health threat posed by these enzymes, novel, effective therapeutics are urgently needed. Here, we report evaluation of a novel, C5α-methyl-substituted carbapenem, NA-1-157, and show that its MICs against bacteria producing OXA-48-type enzymes were reduced by 4- to 32-fold when compared to meropenem. When combined with commercial carbapenems, the potency of NA-1-157 was further enhanced, resulting in target potentiation concentrations ranging from 0.125 to 2 µg/mL. Kinetic studies demonstrated that the compound is poorly hydrolyzed by OXA-48, with a catalytic efficiency 30- to 50-fold lower than those of imipenem and meropenem. Acylation of OXA-48 by NA-1-157 was severely impaired, with a rate 10,000- to 36,000-fold slower when compared to the commercial carbapenems. Docking, molecular dynamics, and structural studies demonstrated that the presence of the C5α-methyl group in NA-1-157 creates steric clashes within the active site, leading to differences in the position and the hydrogen-bonding pattern of the compound, which are incompatible with efficient acylation. This study demonstrates that NA-1-157 is a promising novel carbapenem for treatment of infections caused by OXA-48-producing bacterial pathogens.

The l,d-Transpeptidase LdtAb from Acinetobacter baumannii Is Poorly Inhibited by Carbapenems and Has a Unique Structural Architecture.

l,d-Transpeptidases (LDTs) are enzymes that catalyze reactions essential for biogenesis of the bacterial cell wall, including formation of 3-3 cross-linked peptidoglycan. Unlike the historically well-known bacterial transpeptidases, the penicillin-binding proteins (PBPs), LDTs are resistant to inhibition by the majority of ß-lactam antibiotics, with the exception of carbapenems and penems, allowing bacteria to survive in the presence of these drugs. Here we report characterization of LdtAb from the clinically important pathogen, Acinetobacter baumannii. We show that A. baumannii survives inactivation of LdtAb alone or in combination with PBP1b or PBP2, while simultaneous inactivation of LdtAb and PBP1a is lethal. Minimal inhibitory concentrations (MICs) of all 13 ß-lactam antibiotics tested decreased 2- to 8-fold for the LdtAb deletion mutant, while further decreases were seen for both double mutants, with the largest, synergistic effect observed for the LdtAb + PBP2 deletion mutant. Mass spectrometry experiments showed that LdtAb forms complexes in vitro only with carbapenems. However, the acylation rate of these antibiotics is very slow, with the reaction taking longer than four hours to complete. Our X-ray crystallographic studies revealed that LdtAb has a unique structural architecture and is the only known LDT to have two different peptidoglycan-binding domains.

C6 Hydroxymethyl-Substituted Carbapenem MA-1-206 Inhibits the Major Acinetobacter baumannii Carbapenemase OXA-23 by Impeding Deacylation.

Acinetobacter baumannii has become a major nosocomial pathogen, as it is often multidrug-resistant, which results in infections characterized by high mortality rates. The bacterium achieves high levels of resistance to ß-lactam antibiotics by producing ß-lactamases, enzymes which destroy these valuable agents. Historically, the carbapenem family of ß-lactam antibiotics have been the drugs of choice for treating A. baumannii infections. However, their effectiveness has been significantly diminished due to the pathogen's production of carbapenem-hydrolyzing class D ß-lactamases (CHDLs); thus, new antibiotics and inhibitors of these enzymes are urgently needed. Here, we describe a new carbapenem antibiotic, MA-1-206, in which the canonical C6 hydroxyethyl group has been replaced with hydroxymethyl. The antimicrobial susceptibility studies presented here demonstrated that this compound is more potent than meropenem and imipenem against A. baumannii producing OXA-23, the most prevalent CHDL of this pathogen, and also against strains producing the CHDL OXA-24/40 and the class B metallo-ß-lactamase VIM-2. Our kinetic and mass spectrometry studies revealed that this drug is a reversible inhibitor of OXA-23, where inhibition takes place through a branched pathway. X-ray crystallographic studies, molecular docking, and molecular dynamics simulations of the OXA-23-MA-1-206 complex show that the C6 hydroxymethyl group forms a hydrogen bond with the carboxylated catalytic lysine of OXA-23, effectively preventing deacylation. These results provide a promising strategy for designing a new generation of CHDL-resistant carbapenems to restore their efficacy against deadly A. baumannii infections. IMPORTANCE Carbapenem antibiotics are the drugs of choice for treatment of deadly infections caused by Gram-negative bacteria. However, their efficacy is severely compromised by the wide spread of carbapenem-hydrolyzing class D ß-lactamases (CHDLs). The importance of this research is the discovery that substitution of the canonical hydroxyethyl group of carbapenems by a hydroxymethyl significantly enhances stability against inactivation by the major CHDL of Acinetobacter baumannii, OXA-23. These results provide a novel strategy for designing next-generation, carbapenemase-stable carbapenems to fight multidrug-resistant infections caused by Gram-negative pathogens.

Effects of Inactivation of d,d-Transpeptidases of Acinetobacter baumannii on Bacterial Growth and Susceptibility to ß-Lactam Antibiotics.

Resistance to ß-lactams, the most used antibiotics worldwide, constitutes the major problem for the treatment of bacterial infections. In the nosocomial pathogen Acinetobacter baumannii, ß-lactamase-mediated resistance to the carbapenem family of ß-lactam antibiotics has resulted in the selection and dissemination of multidrug-resistant isolates, which often cause infections characterized by high mortality rates. There is thus an urgent demand for new ß-lactamase-resistant antibiotics that also inhibit their targets, penicillin-binding proteins (PBPs). As some PBPs are indispensable for the biosynthesis of the bacterial cell wall and survival, we evaluated their importance for the growth of A. baumannii by performing gene inactivation studies of d,d-transpeptidase domains of high-molecular-mass (HMM) PBPs individually and in combination with one another. We show that PBP3 is essential for A. baumannii survival, as deletion mutants of this d,d-transpeptidase were not viable. The inactivation of PBP1a resulted in partial cell lysis and retardation of bacterial growth, and these effects were further enhanced by the additional inactivation of PBP2 but not PBP1b. Susceptibility to ß-lactam antibiotics increased 4- to 8-fold for the A. baumannii PBP1a/PBP1b/PBP2 triple mutant and 2- to 4-fold for all remaining mutants. Analysis of the peptidoglycan structure revealed a significant change in the muropeptide composition of the triple mutant and demonstrated that the lack of d,d-transpeptidase activity of PBP1a, PBP1b, and PBP2 is compensated for by an increase in the l,d-transpeptidase-mediated cross-linking activity of LdtJ. Overall, our data showed that in addition to essential PBP3, the simultaneous inhibition of PBP1a and PBP2 or PBPs in combination with LdtJ could represent potential strategies for the design of novel drugs against A. baumannii.

In Crystallo Time-Resolved Interaction of the Clostridioides difficile CDD-1 enzyme with Avibactam Provides New Insights into the Catalytic Mechanism of Class D ß-lactamases.

Class D ß-lactamases have risen to notoriety due to their wide spread in bacterial pathogens, propensity to inactivate clinically important ß-lactam antibiotics, and ability to withstand inhibition by the majority of classical ß-lactamase inhibitors. Understanding the catalytic mechanism of these enzymes is thus vitally important for the development of novel antibiotics and inhibitors active against infections caused by antibiotic-resistant bacteria. Here we report an in crystallo time-resolved study of the interaction of the class D ß-lactamase CDD-1 from Clostridioides difficile with the diazobicyclooctane inhibitor, avibactam. We show that the catalytic carboxylated lysine, a residue that is essential for both acylation and deacylation of ß-lactams, is sequestered within an internal sealed pocket of the enzyme. Time-resolved snapshots generated in this study allowed us to observe decarboxylation of the lysine and movement of CO2 and water molecules through a transient channel formed between the lysine pocket and the substrate binding site facilitated by rotation of the side chain of a conserved leucine residue. These studies provide novel insights on avibactam binding to CDD-1 and into the catalytic mechanism of class D ß-lactamases in general.

Inhibition of the Clostridioides difficile Class D ß-Lactamase CDD-1 by Avibactam.

Avibactam is a potent diazobicyclooctane inhibitor of class A and C ß-lactamases. The inhibitor also exhibits variable activity against some class D enzymes from Gram-negative bacteria; however, its interaction with recently discovered class D ß-lactamases from Gram-positive bacteria has not been studied. Here, we describe microbiological, kinetic, and mass spectrometry studies of the interaction of avibactam with CDD-1, a class D ß-lactamase from the clinically important pathogen Clostridioides difficile, and show that avibactam is a potent irreversible mechanism-based inhibitor of the enzyme. X-ray crystallographic studies at three time-points demonstrate the rapid formation of a stable CDD-1-avibactam acyl-enzyme complex and highlight differences in the anchoring of the inhibitor by class D enzymes from Gram-positive and Gram-negative bacteria.

A surface loop modulates activity of the Bacillus class D ß-lactamases.

The expression of ß-lactamases is a major mechanism of bacterial resistance to the ß-lactam antibiotics. Four molecular classes of ß-lactamases have been described (A, B, C and D), however until recently the class D enzymes were thought to exist only in Gram-negative bacteria. In the last few years, class D enzymes have been discovered in several species of Gram-positive microorganisms, such as Bacillus and Clostridia, and an investigation of their kinetic and structural properties has begun in earnest. Interestingly, it was observed that some species of Bacillus produce two distinct class D ß-lactamases, one highly active and the other with only basal catalytic activity. Analysis of amino acid sequences of active (BPU-1 from Bacillus pumilus) and inactive (BSU-2 from Bacillus subtilis and BAT-2 from Bacillus atrophaeus) enzymes suggests that presence of three additional amino acid residues in one of the surface loops of inefficient ß-lactamases may be responsible for their severely diminished activity. Our structural and docking studies show that the elongated loop of these enzymes severely restricts binding of substrates. Deletion of the three residues from the loops of BSU-2 and BAT-2 ß-lactamases relieves the steric hindrance and results in a significant increase in the catalytic activity of the enzymes. These data show that this surface loop plays an important role in modulation of the catalytic activity of Bacillus class D ß-lactamases.

Structural basis for the diversity of the mechanism of nucleotide hydrolysis by the aminoglycoside-2''-phosphotransferases.

Aminoglycoside phosphotransferases (APHs) are one of three families of aminoglycoside-modifying enzymes that confer high-level resistance to the aminoglycoside antibiotics via enzymatic modification. This has now rendered many clinically important drugs almost obsolete. The APHs specifically phosphorylate hydroxyl groups on the aminoglycosides using a nucleotide triphosphate as the phosphate donor. The APH(2'') family comprises four distinct members, isolated primarily from Enterococcus sp., which vary in their substrate specificities and also in their preference for the phosphate donor (ATP or GTP). The structure of the ternary complex of APH(2'')-IIIa with GDP and kanamycin was solved at 1.34 Šresolution and was compared with substrate-bound structures of APH(2'')-Ia, APH(2'')-IIa and APH(2'')-IVa. In contrast to the case for APH(2'')-Ia, where it was proposed that the enzyme-mediated hydrolysis of GTP is regulated by conformational changes in its N-terminal domain upon GTP binding, APH(2'')-IIa, APH(2'')-IIIa and APH(2'')-IVa show no such regulatory mechanism, primarily owing to structural differences in the N-terminal domains of these enzymes.

The crystal structures of CDD-1, the intrinsic class D ß-lactamase from the pathogenic Gram-positive bacterium Clostridioides difficile, and its complex with cefotaxime.

Class D ß-lactamases, enzymes that degrade ß-lactam antibiotics and are widely spread in Gram-negative bacteria, were for a long time not known in Gram-positive organisms. Recently, these enzymes were identified in various non-pathogenic Bacillus species and subsequently in Clostridioides difficile, a major clinical pathogen associated with high morbidity and mortality rates. Comparison of the BPU-1 enzyme from Bacillus pumilus with the CDD-1 and CDD-2 enzymes from C. difficile demonstrated that the latter enzymes have broadened their substrate profile to efficiently hydrolyze the expanded-spectrum methoxyimino cephalosporins, cefotaxime and ceftriaxone. These two antibiotics are major contributors to the development of C. difficile infection, as they suppress sensitive bacterial microflora in the gut but fail to kill the pathogen which is highly resistant to these drugs. To gain insight into the structural features that contribute to the expansion of the substrate profile of CDD enzymes compared to BPU-1, we solved the crystal structures of CDD-1 and its complex with cefotaxime. Comparison of CDD-1 structures with those of class D enzymes from Gram-negative bacteria showed that in the cefotaxime-CDD-1 complex, the antibiotic is bound in a substantially different mode due to structural differences in the enzymes' active sites. We also found that CDD-1 has a uniquely long Ω-loop when compared to all other class D ß-lactamases. This Ω-loop extension allows it to engage in hydrogen bonding with the acylated cefotaxime, thus providing additional stabilizing interactions with the substrate which could be responsible for the high catalytic activity of the enzyme for expanded-spectrum cephalosporins.

Structural Insights into the Mechanism of Carbapenemase Activity of the OXA-48 ß-Lactamase.

Carbapenem-hydrolyzing class D carbapenemases (CHDLs) are enzymes that produce resistance to the last-resort carbapenem antibiotics, severely compromising the available therapeutic options for the treatment of life-threatening infections. A broad variety of CHDLs, including OXA-23, OXA-24/40, and OXA-58, circulate in Acinetobacter baumannii, while the OXA-48 CHDL is predominant in Enterobacteriaceae Extensive structural studies of A. baumannii enzymes have provided important information regarding their interactions with carbapenems and significantly contributed to the understanding of the mechanism of their carbapenemase activity. However, the interactions between carbapenems and OXA-48 have not yet been elucidated. We determined the X-ray crystal structures of the acyl-enzyme complexes of OXA-48 with four carbapenems, imipenem, meropenem, ertapenem, and doripenem, and compared them with those of known carbapenem complexes of A. baumannii CHDLs. In the A. baumannii enzymes, acylation by carbapenems triggers significant displacement of one of two conserved hydrophobic surface residues, resulting in the formation of a channel for entry of the deacylating water into the active site. We show that such a channel preexists in apo-OXA-48 and that only minor displacement of the conserved hydrophobic surface residues occurs upon the formation of OXA-48 acyl-enzyme intermediates. We also demonstrate that the extensive hydrophobic interactions that occur between a conserved hydrophobic bridge of the A. baumannii CHDLs and the carbapenem tails are lost in OXA-48 in the absence of an equivalent bridge structure. These data highlight significant differences between the interactions of carbapenems with OXA-48 and those with A. baumannii enzymes and provide important insights into the mechanism of carbapenemase activity of the major Enterobacteriaceae CHDL, OXA-48.

Role of the Hydrophobic Bridge in the Carbapenemase Activity of Class D ß-Lactamases.

Class D carbapenemases are enzymes of the utmost clinical importance due to their ability to confer resistance to the last-resort carbapenem antibiotics. We investigated the role of the conserved hydrophobic bridge in the carbapenemase activity of OXA-23, the major carbapenemase of the important pathogen Acinetobacter baumannii We show that substitution of the bridge residue Phe110 affects resistance to meropenem and doripenem and has little effect on MICs of imipenem. The opposite effect was observed upon substitution of the other bridge residue Met221. Complete disruption of the bridge by the F110A/M221A substitution resulted in a significant loss of affinity for doripenem and meropenem and to a lesser extent for imipenem, which is reflected in the reduced MICs of these antibiotics. In the wild-type OXA-23, the pyrrolidine ring of the meropenem tail forms a hydrophobic interaction with Phe110 of the bridge. Similar interactions would ensue with ring-containing doripenem but not with imipenem, which lacks this ring. Our structural studies showed that this interaction with the meropenem tail is missing in the F110A/M221A mutant. These data explain why disruption of the interaction between the enzyme and the carbapenem substrate impacts the affinity and MICs of meropenem and doripenem to a larger degree than those of imipenem. Our structures also show that the bridge directs the acylated carbapenem into a specific tautomeric conformation. However, it is not this conformation but rather the stabilizing interaction between the tail of the antibiotic and the hydrophobic bridge that contributes to the carbapenemase activity of class D ß-lactamases.

Intrinsic Class D ß-Lactamases of Clostridium difficile.

Clostridium difficile is the causative agent of the deadly C. difficile infection. Resistance of the pathogen to ß-lactam antibiotics plays a major role in the development of the disease, but the mechanism of resistance is currently unknown. We discovered that C. difficile encodes class D ß-lactamases, i.e., CDDs, which are intrinsic to this species. We studied two CDD enzymes, CDD-1 and CDD-2, and showed that they display broad-spectrum, high catalytic efficiency against various ß-lactam antibiotics, including penicillins and expanded-spectrum cephalosporins. We demonstrated that the cdd genes are poorly expressed under the control of their own promoters and contribute only partially to the observed resistance to ß-lactams. However, when the cdd1 gene was expressed under the control of efficient promoters in the antibiotic-sensitive Clostridium cochlearium strain, it produced high-level resistance to ß-lactams. Taken together, the results determined in this work demonstrate the existence in C. difficile of intrinsic class D ß-lactamases which constitute a reservoir of highly potent enzymes capable of conferring broad-spectrum, clinically relevant levels of resistance to ß-lactam antibiotics. This discovery is a significant contribution to elucidation of the mechanism(s) of resistance of the clinically important pathogen C. difficile to ß-lactam antibiotics.IMPORTANCEC. difficile is a spore-forming anaerobic bacterium which causes infection of the large intestine with high mortality rates. The C. difficile infection is difficult to prevent and treat, as the pathogen is resistant to many antimicrobial agents. Prolonged use of ß-lactam antibiotics for treatment of various infectious diseases triggers the infection, as these drugs suppress the abundance of protective gut bacteria, allowing the resistant C. difficile bacteria to multiply. While resistance of C. difficile to ß-lactam antibiotics plays the major role in the development of the disease, the mechanism of resistance is unknown. The significance of our research is in the discovery in C. difficile of ß-lactamases, enzymes that destroy ß-lactam antibiotics. These findings ultimately can help to combat deadly C. difficile infections.

A Synthetic Dual Drug Sideromycin Induces Gram-Negative Bacteria To Commit Suicide with a Gram-Positive Antibiotic.

Many antibiotics lack activity against Gram-negative bacteria because they cannot permeate the outer membrane or suffer from efflux and, in the case of ß-lactams, are degraded by ß-lactamases. Herein, we describe the synthesis and studies of a dual drug conjugate (1) of a siderophore linked to a cephalosporin with an attached oxazolidinone. The cephalosporin component of 1 is rapidly hydrolyzed by purified ADC-1 ß-lactamase to release the oxazolidinone. Conjugate 1 is active against clinical isolates of Acinetobacter baumannii as well as strains producing large amounts of ADC-1 ß-lactamase. Overall, the results are consistent with siderophore-mediated active uptake, inherent activity of the delivered dual drug, and in the presence of ß-lactamases, intracellular release of the oxazolidinone upon cleavage of the cephalosporin to allow the freed oxazolidinone to inactivate its target. The ultimate result demonstrates that Gram-positive oxazolidinone antibiotics can be made to be effective against Gram-negative bacteria by ß-lactamase triggered release.

Centralized Networks to Generate Human Body Motions.

We consider continuous-time recurrent neural networks as dynamical models for the simulation of human body motions. These networks consist of a few centers and many satellites connected to them. The centers evolve in time as periodical oscillators with different frequencies. The center states define the satellite neurons' states by a radial basis function (RBF) network. To simulate different motions, we adjust the parameters of the RBF networks. Our network includes a switching module that allows for turning from one motion to another. Simulations show that this model allows us to simulate complicated motions consisting of many different dynamical primitives. We also use the model for learning human body motion from markers' trajectories. We find that center frequencies can be learned from a small number of markers and can be transferred to other markers, such that our technique seems to be capable of correcting for missing information resulting from sparse control marker settings.

Aminoglycoside resistance profile and structural architecture of the aminoglycoside acetyltransferase AAC(6')-Im.

Aminoglycoside 6'-acetyltransferase-Im (AAC(6')-Im) is the closest monofunctional homolog of the AAC(6')-Ie acetyltransferase of the bifunctional enzyme AAC(6')-Ie/APH(2")-Ia. The AAC(6')-Im acetyltransferase confers 4- to 64-fold higher MICs to 4,6-disubstituted aminoglycosides and the 4,5-disubstituted aminoglycoside neomycin than AAC(6')-Ie, yet unlike AAC(6')-Ie, the AAC(6')-Im enzyme does not confer resistance to the atypical aminoglycoside fortimicin. The structure of the kanamycin A complex of AAC(6')-Im shows that the substrate binds in a shallow positively-charged pocket, with the N6' amino group positioned appropriately for an efficient nucleophilic attack on an acetyl-CoA cofactor. The AAC(6')-Ie enzyme binds kanamycin A in a sufficiently different manner to position the N6' group less efficiently, thereby reducing the activity of this enzyme towards the 4,6-disubstituted aminoglycosides. Conversely, docking studies with fortimicin in both acetyltransferases suggest that the atypical aminoglycoside might bind less productively in AAC(6')-Im, thus explaining the lack of resistance to this molecule.

The role of conserved surface hydrophobic residues in the carbapenemase activity of the class D ß-lactamases.

Carbapenem-hydrolyzing class D ß-lactamases (CHDLs) produce resistance to the last-resort carbapenem antibiotics and render these drugs ineffective for the treatment of life-threatening infections. Here, it is shown that among the clinically important CHDLs, OXA-143 produces the highest levels of resistance to carbapenems and has the highest catalytic efficiency against these substrates. Structural data demonstrate that acylated carbapenems entirely fill the active site of CHDLs, leaving no space for water molecules, including the deacylating water. Since the entrance to the active site is obstructed by the acylated antibiotic, the deacylating water molecule must take a different route for entry. It is shown that in OXA-143 the movement of a conserved hydrophobic valine residue on the surface opens a channel to the active site of the enzyme, which would not only allow the exchange of water molecules between the active site and the milieu, but would also create extra space for a water molecule to position itself in the vicinity of the scissile bond of the acyl-enzyme intermediate to perform deacylation. Structural analysis of the OXA-23 carbapenemase shows that in this enzyme movement of the conserved leucine residue, juxtaposed to the valine on the molecular surface, creates a similar channel to the active site. These data strongly suggest that all CHDLs may employ a mechanism whereupon the movement of highly conserved valine or leucine residues would allow a water molecule to access the active site to promote deacylation. It is further demonstrated that the 6α-hydroxyethyl group of the bound carbapenem plays an important role in the stabilization of this channel. The recognition of a universal deacylation mechanism for CHDLs suggests a direction for the future development of inhibitors and novel antibiotics for these enzymes of utmost clinical importance.

Role of the Conserved Disulfide Bridge in Class A Carbapenemases.

Some members of the class A ß-lactamase family are capable of conferring resistance to the last resort antibiotics, carbapenems. A unique structural feature of these clinically important enzymes, collectively referred to as class A carbapenemases, is a disulfide bridge between invariant Cys69 and Cys238 residues. It was proposed that this conserved disulfide bridge is responsible for their carbapenemase activity, but this has not yet been validated. Here we show that disruption of the disulfide bridge in the GES-5 carbapenemase by the C69G substitution results in only minor decreases in the conferred levels of resistance to the carbapenem imipenem and other ß-lactams. Kinetic and circular dichroism experiments with C69G-GES-5 demonstrate that this small drop in antibiotic resistance is due to a decline in the enzyme activity caused by a marginal loss of its thermal stability. The atomic resolution crystal structure of C69G-GES-5 shows that two domains of this disulfide bridge-deficient enzyme are held together by an intensive hydrogen-bonding network. As a result, the protein architecture and imipenem binding mode remain unchanged. In contrast, the corresponding hydrogen-bonding networks in NMCA, SFC-1, and SME-1 carbapenemases are less intensive, and as a consequence, disruption of the disulfide bridge in these enzymes destabilizes them, which causes arrest of bacterial growth. Our results demonstrate that the disulfide bridge is essential for stability but does not play a direct role in the carbapenemase activity of the GES family of ß-lactamases. This would likely apply to all other class A carbapenemases given the high degree of their structural similarity.

Class D ß-lactamases do exist in Gram-positive bacteria.

Production of ß-lactamases of one of four molecular classes (A, B, C and D) is the major mechanism of bacterial resistance to ß-lactams, the largest class of antibiotics, which have saved countless lives since their inception 70 years ago. Although several hundred efficient class D enzymes have been identified in Gram-negative pathogens over the last four decades, none have been reported in Gram-positive bacteria. Here we demonstrate that efficient class D ß-lactamases capable of hydrolyzing a wide array of ß-lactam substrates are widely disseminated in various species of environmental Gram-positive organisms. Class D enzymes of Gram-positive bacteria have a distinct structural architecture and employ a unique substrate-binding mode that is quite different from that of all currently known class A, C and D ß-lactamases. These enzymes thus constitute a previously unknown reservoir of novel antibiotic-resistance enzymes.

Structural Basis for Enhancement of Carbapenemase Activity in the OXA-51 Family of Class D ß-Lactamases.

Class D ß-lactamases of Acinetobacter baumannii are enzymes of the utmost clinical importance, producing resistance to last resort carbapenem antibiotics. Although the OXA-51-like enzymes constitute the largest family of class D ß-lactamases, they are poorly studied and their importance in conferring carbapenem resistance is controversial. We present the detailed microbiological, kinetic, and structural characterization of the eponymous OXA-51 ß-lactamase. Kinetic studies show that OXA-51 has low catalytic efficiency for carbapenems, primarily due to the low affinity of the enzyme for these substrates. Structural studies demonstrate that this low affinity results from the obstruction of the enzyme active site by the side chain of Trp222, which presents a transient steric barrier to an incoming carbapenem substrate. The Trp222Met substitution relieves this steric hindrance and elevates the affinity of the mutant enzyme for carbapenems by 10-fold, significantly increasing the levels of resistance to these antibiotics. The ability of OXA-51 to evolve into a robust carbapenemase as the result of a single amino acid substitution may, in the near future, elevate the ubiquitous enzymes of the OXA-51 family to the status of the most deleterious A. baumannii carbapenemases, with dire clinical consequences.