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The vertical distance measure in arbitrary units indicates the increase in variance when clusters are merged. A principal component analysis reveals the directions of variability in multidimensional space. Principal component 1 PC1 indicates the direction of highest variability, and PC2 is the direction of the next highest variability in a direction perpendicular to PC1. The directions of the principal components are defined by the contributions of the activities with the alternative substrates.

A loading plot demonstrates how the different substrates contribute to the distribution of the data points in relation to the PCs. Bacterial cell lysis was done as follows. The subsequent steps were according to the manufacturer's suggestions. The 16sRNA gene was used as an internal control for all gene copy number determinations. The cells were prepared as described for RNA isolation. Relative optical intensities of the mature enzyme variants to that of the mature evMBL9 were calculated to allow comparison of relative protein levels.

In a recent study 19 , using an approach designated as SIAFE simultaneous incorporation and adjustment of functional elements , MBL activity was successfully introduced into the protein scaffold of human glyoxalase II The engineered enzyme, evMBL8, obtained by structure-based design accompanied by selection, lost its original glyoxalase II activity but acquired the ability to hydrolyze cefotaxime. We further re-examined loop regions of evMBL8 and found Arg in loop 4 pointing toward the interior of the macromolecule.

Moreover, after homology analysis of sequences significantly similar to that of evMBL8, notably residues Gln, Met, and Thr, were conserved in naturally occurring proteins having the same genetic background settings in loop 4.


By examining the original model published by Park et al. The full-length evMBL9, which consists of amino acids and is 24 kDa, comprises a amino acid signal peptide and a mature peptide amino acids, 21 kDa Fig. Capital letters indicate nucleotide bases matching the sequence of the evMBL8 gene. The nucleotide bases encoding the five amino acid substitutions in the evMBL9 gene are underlined , and the corresponding bases in the evMBL8 gene are italicized under the line.

The increases are given as ratios of MIC values determined for the S. Molecular modeling of evMBL8 identified candidate residues participating in substrate binding 19 , which was the basis for the library A design in this study Fig. Four residues Thr, Val, Trp, and Val in loop 1 are part of the binding site. Phe in loop 2, Asn and Lys in loop 4, and Ala in loop 6 were also chosen for mutagenesis, because they are also quite variable in nature.

The benefit of such a strategy has also been shown in previous studies 32 , Library B was constructed by using a random mutagenesis strategy. The coding region for the mature protein amino acid residues 26— was amplified by error prone PCR at a 0. Interrogation of library B allowed us to examine the potential of improving catalytic activity for all residues in the functional parental enzyme.

To combine site-directed mutagenesis and random mutagenesis, library C was constructed by performing error prone PCR on the pooled variants that were isolated from library A showing increased MIC values compared with wild type evMBL9 strain. The mutagenesis rate of the error prone PCR for library C construction was estimated to be 0. Predicted structure of evMBL8 marked with positions selected for mutations. The five mutations introduced into evMBL8 are in blue , and the six positions randomized in library A are in magenta with side chains shown as sticks. To determine whether the expression of the evMBL9 variant was responsible for the increased resistance, resistant colonies were restreaked on new LA plates containing the same concentrations of antibiotics used for selection with or without 2 m m arabinose.

Only for clones where growth was dependent on the presence of arabinose, the plasmids were transformed to new cells followed by determination of the MICs against the same antibiotics as those used in the selection supplemental Table S2. The mutation frequency was calculated as the number of survivors divided by the number of plated cells Table 1. The frequency of mutations that conferred resistance by overexpression of evMBL9 variants was calculated as the frequency of all types of mutations with arabinose multiplied by the two determined ratios weighted frequency in Table 1.

Even though relatively few resistant mutants were successfully isolated from library B, a similar pattern was observed for all three libraries: the relative MIC to penicillin G was increased most up to more than 6- and fold in the libraries A and C, respectively , and the relative MIC to cephalothin, a first generation cephalosporin, was also highly increased up to more than 4- and fold in libraries A and C, respectively.

A indicates the number of mutants tested for arabinose dependence. B indicates the number of arabinose-dependent mutants. C indicates the number of tested arabinose-dependent mutants. D indicates the number of mutants with evMBL9 variants that conferred resistance in wild type background. The values were calculated as the MIC value for resistant transformants expressing the mutant evMBL9 divided by the MIC value for the strain expressing wild type evMBL9 enzyme; in both cases the enzyme was induced with arabinose.

A , resistant transformants obtained from library A. B , resistant transformants obtained from library B. C , resistant transformants obtained from library C. The promoter region, ribosome-binding site, and coding region of the mutated evMBL9 gene were sequenced for the resistant transformants isolated from libraries A, B, and C. No mutations were found in the promoter region or ribosome-binding site of the evMBL9 variants isolated from libraries A or C, but some appeared in several transformants from library B supplemental Table S3.

The expected and observed amino acid substitutions in the six randomized positions for 33 resistant transformants isolated from library A are summarized in Table 2 , and the observed substitutions were ranked at each position according to their frequency of appearance. The statistical significance of the observed frequency of appearance was tested for each of the expected substitutions using the binomial test.

13.3C: Beta-Lactam Antibiotics: Penicillins and Cephalosporins

The expected and observed amino acid AA substitutions in the six randomized positions for the 33 resistant transformants isolated from the library A. All of the amino acid substitutions identified more than once in individual resistant transformants isolated from the three libraries are listed for the positions other than the six randomized ones Table 3. These substitutions are also likely to contribute to the increased resistance.

It is noteworthy that the Stop codon mutation at positions following Ala occurred frequently, which is consistent with the previous observation that Stop codon occurred in high frequency at position A. In addition, four lysine to glutamate mutations at residue and two lysine to glutamate mutations at residue were identified in individually isolated mutants indicating that a negative charge at these two positions is favorable. Nevertheless, a defined structure of the evMBL9 protein, which is currently unavailable, is required to elucidate the functionality of these substitutions. Amino acids substitutions in positions other than the six randomized ones that appeared more than once in the resistant clones isolated from the three libraries.

The position of a given point in the PC diagram is determined by loadings based on the activities with the alternative substrates. These loadings can be plotted together with the PC scores in a biplot, demonstrating the interdependence of the loadings as well as their correlation with the scores. In the biplot, the points have been colored based on the three groups of the dendrogram.

The red cluster represents mutants with generally high MIC values, and it is noteworthy that five of the seven mutants in this cluster derive from library C. In Fig. Loadings close to one another indicate that resistances to the corresponding antibiotics are highly correlated.

Cefaclor and cefoperazone clearly show the lowest correlation among the antibiotics Fig. Multivariate analysis of the resistance profiles of 52 resistant transformants obtained from libraries A, B, and C. The mutants corresponding to each number in the plots are listed in supplemental Table S4.

The mutants are colored on the basis of the three groups found by hierarchical clustering; red color signifies the cluster deviating the most from the remainder, signifying the most marked general resistance profile. B , loadings biplot showing the loadings of the seven antibiotics together with the principal component scores. The coloring of the transformants corresponds to the colors of the clusters in A. In the analysis, the MIC values determined were normalized to unit variance and mean centered for each antibiotic separately. We measured the relative protein levels for 22 resistant transformants, among which 16, 4, and 2 were randomly selected from libraries A, B, and C, respectively.

The results showed that for most transformants, as the resistance level increased, the protein levels were significantly decreased up to more than fold Fig. To examine the contribution of reduced mRNA level to the protein reduction, we determined the mRNA levels for the 22 transformants, and for 21 of them the relative mRNA level was reduced up to fold, which indicated that mRNA was destabilized at the initial stage of the MBL evolution Fig.

To dissect the influence of mutations on the mRNA and protein levels, the relative mRNA level was plotted against the relative protein level for the 22 transformants Fig. This result suggests that protein stability was decreased by some mutations introduced. Because MIC values were increased for these mutants, this indicates that compared with the wild type evMBL9 strain, catalytic activity of the evolved enzymes was increased. The two perpendicular dotted lines mark the values of the wild type.

In H , the relative mRNA level was plotted against the corresponding relative protein level for the 23 strains. The relative mRNA protein levels of resistant transformants. A , the relative protein fold change was calculated as the protein expression level of the resistant transformants divided by that of the parental strain. B , the relative mRNA fold change was calculated as the mRNA expression level of the resistant transformants divided by that of the parental strain. The normalized MIC value was calculated as the relative MIC value divided by the relative protein level for the resistant transformant.

The values are plotted on a logarithmic scale. To study how the broad spectrum evMBL9 could evolve toward increased resistance, three libraries were designed and constructed. We first explored the functional space of evMBL9 variants by the focused library A targeting six residues that were predicted to be involved in substrate binding. Library B was constructed by using a random mutagenesis approach on evMBL9, whereas library C was constructed by using a similar strategy as for library B, but instead of using the parental evMBL9 gene, a pool of resistant evMBL9 variants isolated from the library A was subjected to random mutagenesis.

In this sense library C can be considered to consist of second generation mutants. This finding is in line with the observation in a directed evolution study carried out with a natural MBL that was evolved toward increased resistance against one cephalosporin without sacrificing a broad substrate profile For most mutants the evMBL9 mRNA and protein levels were reduced, despite providing significantly increased resistance in bacteria, suggesting that the catalytic activities of these mutants were increased by several orders of magnitude Fig.

However, we were unable to purify the wild type evMBL9 protein, although several attempts were made using different strategies, indicating a high instability of this engineered protein. Finally, it is unclear why for 21 of the 22 examined mutants the mRNA level was reduced in several cases by 1 to 2 orders of magnitude.

One possibility for libraries A 16 of 22 mutants and C 2 of 22 mutants is that one or several of the six residues Thr, Val, Phe, Asn, Lys, and Ala targeted for mutagenesis are located in a region of the mRNA that is particularly important for its stability. However, this explanation cannot account for the effects seen with mutants from library B 4 of 22 mutants , because the library was generated by random mutagenesis, and at present we cannot explain the reduced mRNA levels in these mutants.

It has been argued that evolution of drug resistance can follow only a limited number of trajectories in sequence space However, we have previously demonstrated that the evolution of intrinsically promiscuous enzymes can circumvent unfavorable mutations by enhancing activities with alternative substrates From the clinical perspective, it would appear that acquisition of enhanced resistance against one particular antibiotic may be accompanied by collateral resistance against alternative drugs. Such evolution of multidrug resistance could jeopardize chemotherapy. However, by means of multivariate analysis of resistance data obtained with different drugs, it is possible to distinguish antibiotics that can be expected to impart the least covariation of resistance.

In addition, by cataloging the substrate specificities of resistance enzymes, as outlined in this study, this type of knowledge could be used in the clinic to tailor antibiotic use and thereby more effectively treat infections caused by resistant pathogens. You'll be in good company. Journal of Lipid Research. Background: Microbial multidrug resistance is a major global problem.

ß-Lactams: Mechanisms of Action and Resistance

Previous Section Next Section. Construction of the S. Library Construction To explore the functional space of evMBL9 variants, three libraries were constructed. Liam Worrall and Robert Gruninger were involved during my training on X-ray crystallography equipment. The manuscripts were edited by all co-authors involved. Versions of chapters 2 and 3 have been published: King D.

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Protein Sci. Strynadka and Dr. Gerard Wright at the University of McMaster. For this paper, I completed all structural work and was responsible for all manuscript preparation. Andrew King, a student in Dr. Wrights lab was responsible for kinetic experiments on mutant enzymes, site-directed mutagenesis, dynamic light scattering, and LC-MS experiments. Manuscript editing was done by all co-authors involved. ACS Infect. DOI: Gerard Wright. Detailed author contributions are as follows: D.

K and D. K performed enzyme kinetics and all MIC experiments; S. B, and D. W principally wrote the manuscript with input from all. A version of chapter 5 has been published: Andrew M. Andrew N. Alexander, Marija Vuckovic, Thomas R. Parr Jr. Strynadka, and Gerard D. ACS Chem. I would like to thank my supervisor Dr. Natalie Strynadka for her mentorship and for providing me with the opportunity to pursue my own research interests and for teaching me countless valuable lessons along the way.

Strynadka has been instrumental in my development as a young scientist particularly in the areas of scientific communication, critical thinking, and manuscript preparation. I would also like to thank my committee members Dr. Lawrence McIntosh and Dr. Filip Van Petegem for their thoughtful insights and critical feedback throughout. I am grateful for past and present lab members who have been key to my development as a structural biologist. In particular, I would like to thank Dr. Susan Safadi for being an excellent mentor, colleague, and friend throughout my first two years in the lab.

I would also like to thank Dr. Liam Worrall, Dr. I would like to thank Dr. Gerard Wright and his student Andrew King for their fruitful collaboration with regards to the avibactam work. Finally, I would like to thank all the members of the Strynadka lab for providing help and support throughout my research. I am also grateful for the scholarship support that I have received from the Canadian Institutes of Health Research.

In Gram-negative bacteria, the PG layer is an essential cell surface feature that sits between the inner and outer membranes and helps to protect the bacterium from osmotic rupture due to membrane turgor pressure and is the defining layer that governs cell shape and morphogenesis 1.

PG biosynthesis initiates with the well-characterized Mur enzyme pathway [reviewed in 1 ], responsible for the synthesis of the UDP-N-acetylmuramic acid MurNAc pentapeptide precursor molecule. This precursor is then attached via a pyrophosphate linkage to the membrane anchored C55 lipid carrier by the integral membrane phosphotransferase MraY. Recently, it has been shown that FtsW is directly involved in the transport of lipid II across the membrane, although the possibility of other candidates performing complementary or redundant roles is not excluded 4.

Secondly, the newly incorporated pentapeptide of the growing chain is cross-linked to a preexisting PG chain by the transpeptidase TP action of PBPs Figure 1. Class A PBPs are bi-functional enzymes harboring both GT and TP activities in distinct catalytic sites, whereas class B PBPs have only TP activity [reviewed in 1 ] and are thought to play more prominent roles during specialized cellular events such as division or in response to environmental cues including antibiotic stress. The head subdomain includes an active site span sufficient for the binding of 6 sugar moieties flanking a conserved catalytic glutamic acid E in the PG-GT family.

The jaw subdomain consists of an inner-helix, an outer-helix and a hydrophobic channel, which together define a putative donor lipid binding site Figure 1. Co-crystallization of S. It is currently unclear whether K, R or E stabilize the pyrophosphate leaving group by direct protonation or coordination of a metal ion 5,7.

Following glycosyl bond formation, a patch of basic residues flanking the donor binding site is proposed to create an electropositive sink for positioning the pyrophosphate of the growing glycan-linked CPP product. Analysis of the distribution of glycan products in a gel electrophoresis assay using 14C labeled lipid II revealed that PG GTases catalyze polymerization in a processive manner, meaning that they undergo multiple successive rounds of glycosyltransfer without releasing the growing polymer 6.

A recent crystal structure of the S. In this structure, R and R form electrostatic interactions with the acceptor pyrophosphate group and are required for lipid II polymerization, as shown by mutagenesis studies Figure 1. A distinct crystal form of partially truncated S. Interestingly, the donor site requires a lipid tether length of at least 20 carbon units whereas the acceptor site displays a broad tolerance of lipid lengths Synthesis of large glycan chains requires the presence of the full transmembrane domain of Streptococcus pneumonia PBP2a, further corroborating the notion that membrane proximity and interaction is a key determinant for processive polymerization Despite having low nanomolar inhibitory activity, moenomycin is impractical for use in humans due largely to poor bioavailability and long serum half-life resulting in poor pharmacokinetic properties However, Kahne and colleagues have recently reported the design and use of an analog containing the minimal moenomycin pharmacophore, linked to a fluorescent probe that was used to screen for low micromolar affinity GT inhibitors Furthermore, Herdewijn and colleagues have synthesized a series of lipid II substrate analogs that display low micromolar affinity inhibition of PBP1b catalyzed glycosyltransfer, and have antibacterial activity against Bacillus subtilis Recently, Cooper and colleagues have used moenomycin and other GT inhibitors as templates to synthesize a novel pyranose scaffold based compound library.

They identify two novel monosaccharides that display in vitro inhibition comparable to moenomycin, with excellent in vivo efficacy in a mouse mammary gland S. Taken together, the rapidly evolving structural understanding of GT mediated lipid II polymerization and improved fluorescence-based assays make the informed design of structure-based inhibitors more tangible than ever. TPs catalyze a two-step reaction that begins with serine-mediated acylation of the position 4 D-alanine carbonyl in the stem-peptide of the growing strand.

This intermediate is then deacylated via nucleophilic attack by a side chain amino group in the 3rd position of the pentapeptide typically meso-DAP or L-lysine-pentaglycine on an adjacent PG strand resulting in transpeptidation [Figure 1. In the TP active site the binding location for the acceptor pentapeptide is as of yet unknown in contrast to that of the donor pentapeptide.

This hypothesis is indirectly supported by the observation that transpeptidation only occurs on a glycan-polymerized substrate 16,17 , hence it is likely that the activity of the TP and GT active sites is in some way coordinated. However, this hypothesis is yet to be directly validated and is complicated by the fact that the PBPs are part of a multi-protein synthase machine and thus the regulation and mechanics of synthesis must be viewed in this context The limitations in our current understanding of PG synthesis are likely in part due to the fact that the building blocks of the PG synthesis machinery have traditionally been studied independently rather than as an integrated macro-molecular machine.

The donor molecule mimic moenomycin bound to PBP2 is depicted as pink sticks with atoms colored by type. The lipid II analog bound S. However, further target identification had to wait 20 years for elucidation of the peptidoglycan PG chemical architecture and biosynthetic pathway [for a detailed review on PG synthesis, please see 1 ]. The bacterial PG is a vast glycan mesh that envelops the entire cell and imparts the rigidity necessary to define cell shape and morphogenesis as well as protect the cell from osmotic rupture The PBP TPs typically catalyze a two-step reaction in which the position 3 amino group of an acceptor strand attacks the peptide bond of the terminal D-alanine-D-alanine of a donor strand, releasing the D-alanine leaving group and forming a peptide cross-link 24, The inhibition of PBPs ultimately results in reduced PG stem peptide cross-links and deregulation of PG degradation, which causes the accumulation of sacculus defects These localized PG defects result in the inability of the cell wall to withstand the osmotic turgor pressure of the cytoplasmic membrane resulting in outer-membrane encased balloon-like structures on the surface of the bacterial cell that eventually rupture leading to cell death This transiently formed intermediate is stabilized by hydrogen bonding to conserved residues in the oxyanion hole comprised of main chain amides of motifs i and iii.

Subsequently, the tetrahedral intermediate collapses to expel the negatively charged nitrogen leaving group which is presumably stabilized by protonation via S motif ii , thereby forming an acyl-enzyme intermediate. Figure 1. The acylated benzylpenicillin is depicted as pink sticks with atoms colored by type. Hydrogen bonding and electrostatic interactions are shown as black dashes. Today, there are four major penicillin subclasses: i natural penicillins, ii penicillinase-resistant penicillins, iii aminopenicillins, and iv extended-spectrum penicillins The evolution of bacterial resistance to natural product penicillins stimulated a renaissance in the development of novel semisynthetic derivatives, which are made using the 6-aminopenicillanic acid 6-APA precursor molecule The cephalosporins the first of which, cephalosporin C, was isolated from the fungi Cephalosporium acremonium in have a six-membered dihydrothiazine ring attached to the lactam core Fig.

Interest in the clinical development of cephalosporins stemmed from their resistance to hydrolysis by penicillinases The side chains used in the development of semisynthetic penicillins were incorporated into the cephalosporin core scaffold However, in contrast to the penicillins, the cephalosporin core offers an additional site of variation at the C3 position Figure 1. The most recent cephalosporins in development either display antipseudomonal activity or are effective against methicillin-resistant Staphylococcus aureus MRSA eg. The carbapenems [the first of which, thienamycin, was discovered in the mids as a metabolic product of Streptomyces cattleya 34 ] have a five-membered 2,3 unsaturated ring with a C1 carbon rather than sulfur 4,5 fused to the lactam core.

Today, carbapenems are often our last line of defense against multidrug-resistant Gram-negative pathogens. However, clinically available carbapenems have low oral bioavailability and thus do not readily penetrate gastrointestinal tissues and are typically administered intravenously Aztreonam is currently the only clinically approved monobactam. Due to its relatively narrow spectrum of activity, aztreonam is generally used as part of antibiotic combination therapies such as aztreonam-vancomycin It is now commonplace for individual bacteria to have multiple different resistance genes that function in concert to confer extended-spectrum resistance.

MRSA and Enterococcus faecium] 46, The two most common mechanisms that regulate this phenomenon at the Gram-negative outer-membrane are the restricted entry of drugs via the alteration or loss of porins and their active expulsion via multi-drug efflux pumps [reviewed in 48,49 ].

Exogenous resistance genes are usually acquired by bacteria through transformation, conjugation, and transduction 51 The new genetic material is then either incorporated into the bacterial chromosome or replicates separately. The mobile genetic elements that facilitate horizontal gene transfer are: i extra-chromosomal double-stranded circular DNA plasmids , ii DNA sequences that can insert themselves into alternate locations in the genome transposons , and iii genetic assembly elements that can capture and incorporate gene cassettes by site-specific recombination integrons [reviewed in 52,53 ].

The class B enzymes are further categorized into the subclasses B1, B2, and B3 based upon amino acid sequence similarities. Today, the class A CTX-M enzymes are the most prominent ESBLs globally and have the ability to readily hydrolyze extended-spectrum cephalosporins such as cefotaxime KPC-2 also commonly located on IncFII plasmids is the most frequently reported class A carbapenemase to date and has been found as the causative agent in numerous carbapenem-resistant nosocomial outbreaks There are currently two proposed mechanisms for S70 activation: i K73 acts as a general base to deprotonate the catalytic S70 60,61 , and ii E activates a water molecule which subsequently deprotonates S70 Subsequently, the tetrahedral intermediate breaks down to expel the N4 nitrogen leaving group, which is protonated by S resulting in the formation of a transient acyl-enzyme intermediate.

K73 is thought to shuttle a proton to S for leaving group protonation during this process However, today many of these enzymes show high catalytic efficiency toward the penicillins These enzymes are typically chromosomally encoded carbapenemases that are often under inducible expression. However, several class C enzymes have now been found localized on high copy number mobile plasmids The class C enzymes are predominantly found in Gram-negative organisms such as E. The class D genes are typically plasmid encoded and are often localized to gene cassettes in integron regions.

Recently, the carbapenem-hydrolyzing OXA enzyme has gained attention due to its broad substrate specificity and large clinical prevalence in carbapenem-resistant Enterobacteriaceae In addition, the motif i K70 is N-carboxylated to a varying extent depending on the particular OXA enzyme under consideration Figure 1. Furthermore, the carboxylated K70 is positioned ideally to activate the deacylating water during hydrolytic deacylation By , only four MBL enzymes had been discovered, and each appeared to be chromosomally encoded and species specific. For the following two decades, the MBLs were seen as interesting, yet clinically insignificant.

However, in , the discovery of plasmid-encoded IMP-1 from P. MBL-mediated resistance in nosocomial infections has gained traction in many multidrug-resistant Gram-negative pathogens including P. Today, MBLs are predominantly plasmid encoded as part of mobile genetic cassettes, which facilitates their transmission throughout microbial populations The blaNDM-1 gene is broadly disseminated in Enterobacteriaceae and is not restricted to a particular plasmid family Additionally, bacteria co-expressing SBLs and MBLs are often capable of hydrolyzing the clinically relevant monobactam aztreonam Despite vast research efforts, and due in part to the lack of a covalently bound adduct during hydrolysis, the development of a clinically useful MBL inhibitor is yet to materialize.

Although MBLs are generally homovalent zinc-dependent hydrolases, several have nevertheless been found to bind cobalt and cadmium in addition to zinc, with varying degrees of hydrolytic efficiency The MBLs are either mono-zinc or di-zinc depending upon the particular enzyme subclass being considered. The B1 and B3 enzymes utilize a di-zinc center to mediate hydrolysis, whereas the B2 MBLs are mono-zinc enzymes that are inhibited by the presence of a second active site zinc ion and display high specificity for carbapenem hydrolysis 84, In the substrate free form of subclass B1 and B3 MBLs, Zn1 is coordinated in a tetrahedral fashion by three universally conserved histidine residues and a bridging hydroxide ion.

Both the subclass B1 and B3 enzymes coordinate Zn2 with trigonal bipyramidal geometry yet differ in the ligands typically utilized. In crystal structures of the subclass B1 and B3 MBLs, Zn1 is typically refined with higher average occupancy than Zn2, suggesting potentially weaker binding of the latter ion. However, catalytic efficiency is generally believed to be optimal when the subclass B1 and B3 enzymes are in the di-zinc form 88, The C3 carboxylate of the substrate forms an electrostatic interaction with the conserved K Figure 1.

Binding by the electropositive zinc ions maintains the bridging catalytic hydroxide at a measured pKa of 5—6 Nucleophilic attack by this hydroxide on the activated carbonyl results in the formation of a tetrahedral intermediate, which is stabilized by a predicted oxyanion hole consisting of Zn1 and potentially the amide side chain nitrogen of N The product is subsequently released from the active site, and the nucleophilic hydroxide is reloaded between the zinc ions for another round of catalysis. For a more complete analysis of the MBL catalytic mechanism, please refer to the following reviews 87, The CTX-M-9 protein is depicted as a green cartoon with selected active site residues shown as pink sticks.

Acylated cefotaxime is depicted as blue sticks. The NDM-1 protein is depicted as a cyan cartoon with selected active site residues shown as beige sticks with atoms colored by type. Hydrolyzed methicillin is depicted as pink sticks. The AmpC protein is depicted in blue cartoon representation with selected active site residues shown as gold sticks with atoms colored by type. Acylated ceftazidime is shown as cyan sticks. The OXA-1 protein is depicted in dark teal cartoon representation with selected active site residues shown as orange sticks with atoms colored by type.

Acylated doripenem is shown as pink sticks. In a—d , a close-up of the native left and acylated right enzyme is depicted. In all panels, hydrogen bonding and electrostatic interactions are shown as blue dashes, and all non-carbon ligand and residue atoms are colored by type O; red, N; blue, S; yellow. These inhibitors form a long-lived acyl-enzyme intermediate with the catalytic serine, characterized by a very slow rate of hydrolytic deacylation. Following acylation, a second ring-opening event occurs leading to a stable imine that undergoes various chemical transformations.

Eventually, the acyl-enzyme hydrolytically deacylates either directly, or through a series of covalent intermediates to yield active enzyme and inactivated product Typically, new inhibitors are paired with either late generation cephalosporins or carbapenems in large part due to their broad-spectrum activity.

MK is structurally very similar to avibactam with the addition of a piperidine ring attached to the C2 carboxamide hereafter referred to as R1 Figure 1. The role of cilastatin is to prolong the half-life of imipenem by inactivating human dehydropeptidase, which would otherwise readily degrade the carbapenem.

Boronic acid SBL inhibitors Boron contains an empty p-shell making it an excellent electrophile with a high propensity to form dative covalent bonds with active site serine nucleophiles , Since their initial discovery in the late s, the boronates have proven to be effective SBL inhibitors in vitro.

A novel heterocyclic boronate inhibitor RPX is being developed by Rempex Pharmaceuticals and displays strong potentiation of the carbapenem antibiotic biapenem against class A carbapenemase producing Enterobacteriaceae RPX contains a thiophene moiety in an analogous position to the R1 group of the nitrocefin and cefoxitin cephalosporins Figure 1.

A major challenge for the future will be the design of clinically useful boronic acid inhibitors that target the class B and D enzymes. A major consideration in development of the boronic acid SBL inhibitors is their propensity for off-target inhibition of serine proteases and the proteasome, such that structure-guided functionalization conferring target specificity is of prime importance in their continued development Most notably however is that in mice infected with NDM-1 positive Klebsiella pneumoniae, AMA was able to effectively restore meropenem activity , Recently, Brem and colleagues characterized rhodanine hydrolysis products as potent MBL inhibitors and show that they complex with the VIM-2 active site zinc ions via thioenolate mediated zinc intercalation The class D OXA SBLs, were originally named for their ability to hydrolyze oxacillin and are a diverse group of enzymes with substrate hydrolysis profiles spanning from narrow to broad.

Also of note is the encouraging ability of avibactam to inhibit certain class D enzymes. The observed variability is predominantly due to discrepancies in carbamylation rather than decarbamylation rates, an attribute that should be considered in future DBO drug design efforts Identifying new compounds and chemical strategies to address the resistance issue is paramount. Chapter 2 describes the structural and biochemical characterization of K.

Crystallographic analysis of holo-NDM-1 as well as oligomerization and localization data is presented. This work has been published in Protein Science Chapter 3 describes the structural characterization of multiple ligands bound to NDM The high-resolution X-ray crystal structures of NDM-1 bound to the hydrolyzed penicillin antibiotics benzyl penicillin, oxacillin and methicillin as well as the hydrolyzed carbapenem meropenem and high affinity MBL inhibitor L-captopril are provided. This work has been published in the Journal of the American Chemical Society Chapter 5 involves the characterization of a novel series of diazabicyclooctane avibactam derivatives.

We find that the diazabicyclooctane derivatives act as potent antimicrobial agents outright and display robust antibacterial activity against clinical isolates of P. Furthermore, we use E. Specifically, they attenuate peptidoglycan biosynthesis by inhibiting the transpeptidase enzymes, involved in cross linking adjacent peptidoglycan strands via their penta-peptide repeats This strategy for antibiotic treatment has been very successful due largely to limited toxicity, excellent bioavailability and broad activity Classes A, C and D use an active site serine as a nucleophile.

King D. However, despite vast research efforts in this area, there are currently no effective inhibitors targeted against the emerging class B enzymes. MBL enzymes are now found widely disseminated throughout the world Coupled with their broad-spectrum substrate profile the clinical threat of MBLs is increasingly dire. This novel resistance gene was discovered in India and has rapidly spread throughout human populations on nearly every continent NDM-1 positive Escherichia coli are now widespread in the environment and water supplies in India , likely a result of the fact that NDM-1 genes are typically located on readily transferable plasmids that are prone to rearrangement Due to the selective advantage that NDM-1 confers and its propensity for plasmid-mediated horizontal gene transfer, it is feared that NDM-1 may herald the end of treatment with those drugs that are used clinically to fight Gram-negative infections , In order to gain a more complete understanding of the structural basis for the antibiotic resistance conferred by NDM-1, we have determined the crystal structure of the holo form of this enzyme to a resolution of 2.

Comparison of our NDM-1 structure with the very recent product complex form of NDM-1 as well as other characterized Class B MBLs, provides important new insights into the catalytic mechanism within the active site as well as unique molecular features that may contribute to its broad spectrum antibiotic specificity. NDM-1 protein was expressed in E. Elution buffer 50 mM Tris pH7,. Crystal conditions were. Simulated annealing was performed on the partially refined model using Phenix In the final stages of refinement, NCS restraints were relaxed and TLS groups residues , and were defined to allow for differences in loop regions between the five monomers.

Zinc and waters were added manually by examination of Fo-Fc and 2Fo-Fc maps and refined at full occupancy. Lysate was centrifuged at 9, rpm for 15 min followed by centrifugation 45,rpm in a Beckman 60 Ti rotor for 1hr to isolate cell membranes. Prior to sample analysis, the ICP-MS was calibrated using a standard solution containing the metal isotopes of interest Inorganic Ventures.

Instrument settings were: rf power W , integration time 35s , collision gas Ar40 , RPQ voltage 25V and sample flow rate 4 rpm. Isotope abundance was determined by integrating peak areas using the NexION software program, and the data was represented graphically using Prism. The light source for the system was a mW air launched laser, operating at nm. The structure of holo-NDM-1 was solved by molecular replacement phasing and subsequently refined to a resolution of 2. The crystals are P1 with five protein chains in the asymmetric unit, and a Matthews coefficient of 2.

The final refined model has an Rwork and Rfree of Each monomer had one residue D90 in a disallowed region on the Ramachandran plot.

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Figure 2. A, Cartoon of holo-NDM-1 structure green , active site zinc ions grey spheres , zinc ligands stick, cpk coloring and active site loops L3 and L10 red. Secondary structure designations are labeled. Negative control is E. Positive control is outer membrane protein ZirT The loop L8 that is unique to NDM-1 forms key contacts involved in this dimer interface.

This suggests a potential functional role in dimerization for this unique insertion. Supporting a potential physiological role for the dimer interface in our crystallographic data, we used size exclusion chromatography, dynamic light scattering and chemical cross linking to show that the M1-R NDM-1 FL enzyme exists as a dimer in solution Figure 2. To our knowledge, the only other example of a class B MBL oligomer is the L1 functional tetramer , This dimerization of purified NDM-1 may be an important feature for antibiotic resistance in a biological context.

Our biochemical analysis also suggests an unusual lipidation and outer membrane localization for NDM Normally, MBL enzymes contain a type I signal peptide. However, the LipoP server revealed probability scores of -. The corresponding peaks are marked, along with appropriate positions of molecular mass standards 67 and 47 kDa. The monomer and dimer fractions, collected from gel filtration are shown in red and blue. The plot indicates that NDM-1 can exist as a dimer in solution.

The results are displayed as particle size distributions with the inset showing the results summary table. Samples were incubated with and without 2. This zippering effect pulls the tip of the L3 loop further away from the zinc center, causing the side chain sulfur of M67 to reorient away from the zinc center by 4. Together, residues L65, M67 and W93 form a hydrophobic face that interacts with the R1 phenyl group of the substrate.

The observed flexibility in the holo-enzyme may be a mechanistic feature of MBLs that provides the L3 loop with the plasticity required to form substrate-specific hydrophobic interactions. Upon ligand binding, the L10 loop also plays an important role in interacting with the substrate. In particular, the N side-chain nitrogen is pulled 1. This shift is larger than the rms differences in superposition of the common CA atoms in the two models of 0. This movement positions the highly conserved N for interaction with the lactam carbonyl group observed in the product complex structure This conserved N residue, together with Zn1 are thought to provide an oxy-anion hole, which helps to polarize the lactam carbonyl upon binding and facilitate nucleophilic attack by the adjacent hydroxide see below The observed plasticity in the L3 and L10 loops likely provide NDM-1 with the ability to accommodate multiple substrates with differing molecular architectures.

It has been proposed that VIM-2 and VIM-4 are restricted in their substrate profile due to the presence of a narrow active site cleft The larger surface area of NDM-1 arises in part from the orientation of the active site loops L3 and L10, which are positioned further away from the zinc active site center see also below when compared to VIM-2 Figure 2.

The protrusion of VIM-2 R into the active site cleft reduces the size of the binding pocket, potentially limiting the VIM-2 substrate binding profile. In addition, VIM-2 has several residues including F61, D62, Y67 and R which project into the active site cleft adding both charge and steric bulk. In equivalent positions, NDM-1 contains residues M67, P68, V73 and A, which present more hydrophobicity in the L3 loop and less steric bulk in the active site Figure 2.

These active site characteristics likely decrease steric interference with incoming substrates and facilitate a favorable electrostatic environment leading to a broad substrate profile. Coordination interactions are blue dashes and hAMP is shown in cpk colored stick representation carbons are magenta.

In addition, the presence of zinc was confirmed by ICP mass spectrometry Figure 2. In this mechanism, Zn1 functions to orientate the substrate carbonyl bond, whereas Zn2 is required for interaction with the amide nitrogen of the substrate A presumed active site hydroxide located between the two zinc ions, which is present in all five holo-NDM-1 monomers and hydrolyzed ampicillin bound NDM-1 , serves as a nucleophile Following attack of the hydroxide on the carbonyl carbon, the peptide bond is broken with Zn2 acting as a Lewis acid to stabilize the charge on the nitrogen leaving group generated during this step Our refined crystallographic data also suggests a consistent differential occupancy of the 2 zinc ions in each of the 5 active sites of the asymmetric unit, in that Zn1 refines with lower average temperature factors B factor For SPM-1, an increased Zn2 coordination distance by the conserved aspartic acid ligand was associated with the lower occupancy metal binding In particular, K, which immediately precedes the Zn2 ligand D presents a positive charge in close proximity to the Zn2 binding site Figure 2.

Therefore, this charge may in part lead to a repulsion of Zn2. The larger Zn-Zn distance observed in the hydrolyzed ampicillin form of NDM-1 likely occurs to accommodate zinc binding to the newly formed carboxylic acid following lactam hydrolysis. This shift exemplifies the role of plasticity, and active site rearrangements in facilitating turnover in MBLs. Taken together, these amino acids may point to an evolutionary trend in promoting broad-spectrum antibiotic resistance.

These antibacterials inhibit cell wall biosynthesis by acting as substrate analogs to prevent the transpeptidase mediated cross-linking of adjacent peptidoglycan strands The prospect that bacteria can develop or acquire high levels of resistance to these and other antibiotics is of global health concern. The class B enzymes are further divided into subclasses B1, B2 and B3 of which the class B1 enzymes have emerged as the most clinically significant and are characterized as having two active site zinc ions Figure 3.

Figure 3. Among the most promising candidates are the L- and D- diasteriomers of the mercaptocarboxamide inhibitor captopril, which display potent and broad-spectrum MBL inhibitory activity Additionally, NDM-1 is encoded on a readily transferrable plasmids, which facilitates its transmission NDM-1 has spread to nearly every continent worldwide and has become a formidable threat to human health, prompting the World Health Organization to issue a global warning , Drops were then streak seeded with finely crushed ampicillin bound NDM-1 crystals prepared as previously described The benzylpenicillin, oxacillin and methicillin bound crystals diffracted to 1.

Meropenem bound crystals diffracted to 1. Ethylene glycol and L-captopril bound crystals diffracted to 1. However, hydrolyzed benzyl penicillin, hydrolyzed meropenem and L-captopril bound structures were refined with isotropic B-factors. Zinc, water and the appropriate ligands were added manually by examination of the Fo-Fc and 2Fo-Fc electron density maps. Figures 3.

The penicillin and meropenem product complexes crystallized in the space groups P and P with two protein chains in the asymmetric unit Table 3. The two chains in the asymmetric unit displayed high structural similarity RMSDs for all common CA atoms in chains A and B for hydrolyzed methicillin, benzylpenicillin, oxacillin and meropenem bound NDM-1; 0. A Hydrolyzed penicillin and hydrolyzed meropenem hMER structures. B Stereoview active site close-up of overlay of penicillin product complexes bound to NDM The NDM-1 structure is represented as a green cartoon and zinc coordinating ligands are green sticks with atoms colored by type.

NDM-1 is shown in green cartoon representation with selected active site residues displayed as sticks and atoms are colored by type. The hMER ligand is slate with atoms colored by type and the 2Fo-Fc electron density is contoured to 0. Zinc ions are shown as grey spheres. Bonds representing zinc coordination, hydrogen bonding, hydrophobic and electrostatic interactions are displayed as thin blue, thin black, thick grey and thick purple dashes. The observed position of the presumed nucleophilic hydroxide, W1, is common to all penicillin product complex structures and is located directly between Zn1 and Zn2 at distances of 2.

Previously, it was thought that following product release W1 is re-loaded between the zinc ions for another round of hydrolysis 90, However, the presence of the nucleophilic W1 in our product complexes suggests that the nucleophilic hydroxide may be re-loaded into its catalytic position even before the product leaves the active site. Atoms within this penicillin core displayed lower average temperature factors than those within the side R1 functional group when averaged across all hydrolyzed products 9.

Thus, the penicillin core coordinates to the NDM-1 zinc center in a precise and rigid conformation, which is independent of the constituents present on the R1 functional group. Remarkably, we see that the enlarged NDM-1 active site cleft easily accommodates the bulky methicillin and oxacillin R1 groups located directly between the L3 and L10 loops Figure 3.

The hOX bound NDM-1 cartoon is green with selected active site residues colored by atom and zinc ions displayed as grey spheres. The hOX ligand is brown with atoms colored by type. The hMETH ligand is slate with atoms colored by type. In all structures, Zn1 is tightly bound at 2. Zn2 displays tight binding to N4 and the C3 carboxylate oxygen at 2.

Our high-resolution penicillin product complex structures in NDM-1 display a particularly strong coordination interaction between the C3 carboxylate and Zn2 Figure 3. Therefore, development of the monobactams presents a logical avenue for the design of novel antibiotics that are not recognized by MBLs.

NDM-1 is shown in green cartoon representation with zinc ions as grey spheres. The hMETH zinc coordinating interactions are shown as blue dashed lines. Indeed, the carbapenem core refines with lower average temperature factors than does the DMP group This observed disorder may be in part responsible for the higher Km values of meropenem for NDM-1 than for either benzylpenicillin or ampicillin However, the large NDM-1 active site cleft provides steric accommodation of the bulky DMP functional group despite the lack of specific interactions, leading to recognition and hydrolysis of meropenem.

The carbapenem and penicillin cores bind differentially to the NDM-1 di-zinc center. The carbapenem N4 and C3 carboxylate oxygen coordinate to Zn2 at 2. However, the newly formed C6 carboxylate directly intercalates Zn1 and Zn2 at 2. This differs from the hydrolyzed penicillin bound structures in which the C6 carboxylate is shifted away from Zn2 toward the L10 loop resulting in a penta rather than hexa coordinated Zn2 Figure 3. The inter-zinc distance is also shorter in the meropenem bound structure, 4. Despite these conformational differences, NDM-1 mediated hydrolysis of meropenem, benzylpenicillin and ampicillin display remarkably similar Kcat values 12s-1, 11s-1 and 15s-1 The observed variability in binding of the penicillin and carbapenem cores in NDM-1 exemplifies the ability of zinc to facilitate multiple ligand geometries and coordination numbers In the meropenem product complex structure, the C6 carboxylate oxygen O71 displays tight hydrogen bonding, 2.

These results provide evidence that N interacts directly with the substrate and thus supports the potential role of this residue in stabilizing the transient tetrahedral intermediate via formation, along with Zn1, of an oxyanion hole. However, we present the first reported crystal structure of an L-captopril inhibited MBL enzyme complex to 2. The two chains displayed high overall structural similarity RMSD 0. The L-captopril S1 intercalates directly between Zn1 and Zn2 at 2. Upon binding, the S1 sulfur displaces the nucleophilic W1 leading to a competitively inhibited enzyme. In this complex both Zn1 and Zn2 display tetrahedral coordination geometries in which the L-captopril S1 forms the final coordination site for each ion Figure 3.

The intercalated S1 presumably decreases electrostatic repulsion between the two zinc ions. L-captopril presents two chemically distinct binding faces, one hydrophobic face which interacts with the L3 loop and the other hydrophilic, which hydrogen bonds to N on the L10 loop. N makes a hydrogen-bond interaction between its backbone amide and the L-captopril O2 at 3.


A Structure of L-captopril C8 chiral carbon is highlighted with a red asterisk. The NDM-1 backbone is represented in green cartoon and selected active site residues are shown as sticks with atoms colored by type. The L-captopril ligand is brown with atoms colored by type and the 2Fo-Fc electron density map is contoured to 1. Hydrogen bonds, zinc coordination bonds and hydrophobic interactions are shown as thin black, thin blue and thick grey dashes.

On this basis, it has been suggested that D-captopril likely adopts the same general orientation within the BlaB and NDM-1 active sites Indeed, NDM-1 contains many potential hydrogen bond donors and acceptors capable of making productive contacts with D-captopril in the BlaB type orientation Figure 3. The fact that both diasteriomers are capable of making productive contacts and display virtually identical S1 zinc intercalation suggests that a fusion of the two molecules whereby D- and L-captopril share a common S1 and C1 would likely generate an even tighter binding inhibitor.

The L-captopril bound NDM-1 protein is shown as a green cartoon and selected active site residues are green sticks, which are colored by atom type. L-captopril, D-captopril and ethylene glycol are brown, light blue and teal with atoms colored by type.

Penicillin-like antibiotics

Proposed D-captopril hydrogen bonding and zinc coordination interactions are displayed as black and blue dashes. Despite having relatively low sequence identity, the class B1 MBLs in general display remarkable structural similarity, especially with respect to the disposition of the catalytic residues and ions Consequently, inhibitory compounds often act universally on all MBLs. Sulfhydryl mediated zinc intercalation is emerging as an exciting avenue for the development of competitive MBL inhibitors , Our product complex structures reveal that the traditional strategy of developing newly functionalized R1 and R2 groups may be readily thwarted by the plastic and open nature of the MBL active site.

These enzymes are often encoded on readily transferable plasmids that facilitate their transmission throughout microbial populations These compounds are mechanism based, covalent inactivators that form a stable acyl-enzyme intermediate with the catalytic serine. At clinically used concentrations, the bound inhibitor undergoes slow hydrolytic deacylation, either directly or through a series of covalent intermediates whereby the heteroatom at the inhibitor 1-position acts as a leaving group and generates an acyclic acyl-enzyme complex that tautomerizes between a stable imine and enamine intermediate The DBO compound avibactam is currently in phase III clinical development as part of a combination therapy in conjunction with ceftazidime to treat complicated urinary-tract and intra-abdominal infections Ceftazidime-avibactam is active against the majority of Enterobacteriaceae, including multi-drug resistant strains, and importantly is effective against Pseudomonas aeruginosa In animal models, avibactam-ceftazidime has been utilized to effectively treat ceftazidime-resistant Gram-negative bacterial septicemia, meningitis, pneumonia and pyelonephritis.

The avibactam-ceftazidime safety and tolerability in clinical trials has been outstanding, and there have been relatively few adverse drug effects documented Although avibactam displays excellent inhibitory activity against the class A and C enzymes, more variable levels of inhibition have been observed towards the class D SBLs Instead decarbamylation of avibactam occurs via recyclization of the DBO fused ring structure, re-forming the intact inhibitor that can then either re-carbamylate the same active site or be released into solution to inactivate subsequent SBLs However, the mechanism and roles of individual amino acids in SBLs that contribute to avibactam activity remain largely unresolved.

To address the underlying molecular details of avibactam inhibition, we have undertaken a multifaceted structural, kinetic and mutagenesis study on known targets.

  • Containing the Beta-Lactam Structure;
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The universally conserved S acts both as a general acid during carbamylation and a general base during de-carbamylation in the avibactam recycling pathway. We further reveal the 2. The data elucidates the active site features likely responsible for the variable inhibition observed for this class of SBL enzymes that uniquely relies on a post-translationally modified carboxylated lysine during catalysis. Genes were synthesized by IDT. For kinetics studies, an E. Lysate was centrifuged using a Beckman JA The column was washed with 5 column volumes of the same buffer and step gradients of increasing imidazole were used for wash and elution steps.

Nitrocefin was synthesized as reported previously Rates of hydrolysis were measured in well microplate format at nm using a Spectramax reader Molecular Dynamics. Absorbance was read continuously at nm. For data analysis, the offset between reaction initiation and the first absorbance read was ms. Data were fit to a two-step reversible inhibition model as described previously , Eq. Error values reported are the standard errors of the fit. Since the samples were incubated in the absence of nitrocefin, the concentration of substrate used in calculations was 0.

After desalting via PD column 1. Data were fit to Equation 4 to obtain koff. In the off-rate experiment, vs was approximated by a no inhibitor control and v0 by a no enzyme control. Error values reported are the standard deviation of three technical replicates. After removal of excess avibactam via PD column 1. Data were fit to Equation 6 to obtain koff At the CLS and x-ray home source, data was collected at a wavelength of 1. All structures were refined with isotropic B-factors. Water and avibactam were added manually by examination of the Fo-Fc and 2Fo-Fc electron density maps.

Figures 4. Size distribution of the samples was calculated based on the correlation function provided by the Zetasizer Nano S software. The P. The expression vectors were then transformed into E. The supernatant was then filtered using a 0. Elution buffer 50 mM Tris, pH 7. Samples were then exchanged via a 10 kDa cut-off Amicon centrifugation concentrator into crystallization buffer 20mM Tris, pH 7. Recently, Docquier et al. Despite this difference in avibactam binding we find that the compound itself takes on a nearly identical conformation between the two crystal forms and resulting structures see Figure B.

Figure 4. Stereoview active site close-up of carbamyl-avibactam bound CTX-M The carbon atoms of avibactam are pink and all other atoms are colored by type N, blue; O, red; S, yellow. The avibactam bound CTX-M protein backbone is displayed as a green cartoon. The catalytic water W1 is shown as a green sphere. Hydrogen bonding and electrostatic interactions are depicted as black dashes. The avibactam sulfate projects into an electropositive pocket formed by K and is stabilized by interactions with T and K on motif iii.

The R1 carboxamide is oriented away from the active site core and is within hydrogen bonding distance 3. We suggest that a major contributing factor to the avoidance of the carbamyl-enzyme to hydrolytic decarbamylation is that the carbamyl carbon is less susceptible to nucleophilic attack than its ester acyl-enzyme counterpart , Carbamyl bond formation is a common feature observed for inhibitors of serine hydrolases, some of which have also been shown to retain the catalytic water in the carbamylated state , To evaluate the mechanistic details governing the reversible recyclization reaction of avibactam inhibition, we mutagenized key active site residues within CTX-M and measured enzyme activity using the colorimetric cephalosporin, nitrocefin, as a reporter substrate.

We confirmed that mutants adopt a WT particle size distribution by dynamic light scattering, and that no hydrolysis of avibactam occurred using liquid chromatography-mass spectrometry LC-MS Figures 4. CTX-M variants show a nearly identical elution profile to WT suggesting they are similarly folded. The second proposal involves K73 as the base responsible for S70 activation.

In contrast, K73A was almost completely carbamylation deficient 2. These data support the proposition that K73 is the general base responsible for S70 activation during avibactam carbamylation. During carbamylation, the avibactam N6 nitrogen is protonated following ring opening. The carbamylation and decarbamylation rates were measured using the colorimetric reporter substrate nitrocefin. The KD values are calculated from the carbamylation and decarbamylation rates.

Despite intensive research on SBLs over the past several decades, this is the first evidence of S acting as a general base, a feature that exemplifies the novelty of the avibactam inhibitor and the functional plasticity of this residue. However, KA only displays a moderate reduction in decarbamylation rate as compared to the wild-type enzyme Table 4. However, for avibactam re-cyclization, we find that the EQ decarbamylation rate is virtually identical to wild-type suggesting that E is not required for S70 protonation Table 4.

In the carbamyl avibactam-CTX-M crystal structure, the side chain amide nitrogen atoms of N and N are within hydrogen bonding distance 2. We found that mutant NA, but not NA, had a 4-fold reduction in carbamylation rate as compared to the WT enzyme but minimal effect on decarbamylation Table 4. Future drug design efforts should seek to maintain a hydrogen bond acceptor at the avibactam C2 carboxamide oxygen position. Therefore, we limit our analysis to chain A for each product complex. All avibactam-bound complexes display clear, and unambiguous ligand omit map Fo-Fc electron density for avibactam within the active site of each protein chain in the ASU Figure 4.

In a-c, the Fo-Fc ligand omit maps are contoured at 3. In all panels, the carbamyl-avibactam is represented as pink sticks with atoms colored by type. The 2Fo-Fc electron density map is contoured at 1. The high resolution of the models allows us to make detailed observations about key active site interactions. From a structural standpoint, the location of S equivalent to S in the class A enzymes , likely also contributes to the observed bond fission as it is ideally positioned to protonate an N6 rather than N1 leaving group Figure 4.

The carbon atoms of avibactam are pink with all other non-carbon atoms colored by atom type.

The avibactam bound OXA protein chain is displayed in orange cartoon representation, and key active site residues are shown as sticks with atoms colored by type. The avibactam bound and unbound OXA protein chains are illustrated in cyan and white cartoon representation, and key active site residues are depicted as sticks with atoms colored by type OXA numbering. The carbamyl-avibactam and OXA protein chain are displayed as in A.

The unbound OXA protein chain is illustrated in grey cartoon representation, and key active site residues are depicted as sticks with atoms colored by type. The carbamyl-avibactam bound structure is displayed as in a. The OXA protein backbone is illustrated as a white cartoon, and key active site residues are shown in stick representation with atoms colored by type. The acyl-oxacillin carbon atoms are grey and all other atoms are colored by type.

In a, b, c, d and f, all hydrogen bonding and electrostatic interactions are depicted as black dashes. In the OXA and OXA carbamylated form, the six membered piperidine ring of avibactam adopts a chair-type conformation, with the C4 and N1 atoms located above and below the plane Figure 4. The C7 carbonyl oxygen of the newly formed carbamyl linkage is located in the oxyanion hole of the enzyme, and is bound by the backbone amide protons of S70 and Y at 2. When aligning the native and avibactam bound OXA and OXA crystal structures, we observe that the protein chains are nearly identical r.

Thus, the class D SBL active site is poised for interaction with avibactam without the need for complicated conformational rearrangements that can substantially slow acylation, as observed for S. Although carboxylation of K73 may be important to the observed carbamylation rates see discussion below , structural differences between enzymes may also play a role. When overlaying avibactam-bound OXA onto the 2. Furthermore, the oxacillin and avibactam ligands display analogous overall orientations despite their chemical differences Figure 4.

The electronegative substituent the avibactam N6 sulfate and penicillin C3 carboxylate both project toward the basic patch defined by R, K and T However, both the avibactam N6 and the analogous oxacillin N4 atoms are within hydrogen bonding distance 3. The N-carboxylation state of K73 is sensitive to pH, with carboxylation increasing at higher pH values presumably due to an increased reactivity of K73 to carbon dioxide in more basic solutions, and the greater stability of carbamic acid in the anionic form at higher pH OXA with no avibactam present was crystallized at pH 7.

Interestingly, in the avibactam bound OXA structures at both pH 6. Therefore, the presence of the bound avibactam appears to disfavor K73 carboxylation in the carbamyl-enzyme complexes. Only in the pH 8. At the same time, these are the only chains from all OXA structures that display partial rather than full occupancy for avibactam Table 4. However, it should be noted, at pH 8. As an interesting aside, to our knowledge, this is the first report showing that avibactam is not stable at high pH. Recently, Ehmann et al. Taken together, the absence of K73 N-carboxylation clearly disfavors hydrolytic decarbamylation of avibactam for the class D enzymes.

However, it is currently unclear whether or not carboxylated-K73 is the general base responsible for S70 activation during carbamylation and how this modification affects avibactam carbamylation rates for the class D SBLs. In the avibactam-bound OXA structure, the de-carboxylated K73 is oriented toward S, rather than W57 as observed in the unbound, carboxylated state Figure 4.

In the avibactam bound form, the de-carboxylated K73 hydrogen bonds to S at a distance of 2. We propose that upon avibactam recyclization, this hydrogen bonding network results in K73 mediated deprotonation of S, which in turn removes the avibactam N6 hydrogen facilitating attack on the carbamyl bond and subsequent re-cyclization. Taken together, these crystal structures along with the CTX-M mutant kinetic data allows us to propose a universal mechanism for SBL inhibition.

Electrostatic stabilization of the N6 sulfate likely helps to orient the bound avibactam in the pre-catalytic Michaelis complex, whereby the sulfate occupies the electropositive pocket formed by the SBL motif iii. The lone pair of electrons on the C7 oxygen drive back into carbonyl formation expelling the negatively charged N6 nitrogen, which is concomitantly protonated by the motif ii serine or tyrosine for the class C enzymes , resulting in the formation of a stable carbamyl enzyme complex.

Upon eventual avibactam decarbamylation, a reversible mechanism of recyclization generally occurs, whereby the SXXK lysine takes part in a concerted acid-base shuffling of protons from the motif ii serine for class A and D , or tyrosine for class C , which deprotonates the N6 nitrogen facilitating an intramolecular nucleophilic attack on the electrophilic C7 carbamyl carbon. Avibactam is a reversible, covalent inhibitor possessing a novel diazabicyclooctane DBO core scaffold recently approved by the U.

Food and Drug Administration FDA in combination with ceftazidime Avicaz to address the challenge of antibiotic resistance Avibactam forms a long-lived carbamyl-linkage with the SBL active site serine. Andrew M. Nitrocefin was synthesized as described previously Vectors containing the E.

The cell lysate was then centrifuged twice at 11, rpm for 15 minutes using a Beckman JA The supernatant was then centrifuged at 45, rpm for 1 hour using a Beckman 60Ti rotor in order to pellet the membranes. The solubilized protein was then purified using nickel chelation chromatography. BL21 DE3 host cells transformed with the E.

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The cell lysate was then centrifuged at 45, rpm for 1 hour using a Beckman 70Ti rotor. The supernatant was purified using nickel chelation chromatography. The column was pre-incubated in the presence of equilibration buffer 50mM Tris pH 8. Peak fractions containing purified protein were pooled and exchanged into assay buffer equilibration buffer , using a 50 KDa cutoff Amicon centrifugal concentrator.

Co-crystallization attempts with FPI and OXA failed to yield suitable crystals so apo crystals were grown as above but without ligand and soaked in 9. All crystallographic data in this study was collected at a temperature of K and wavelength of 1. Data were processed using Xia2 Several iterations of manual rebuilding in Coot , followed by refinement using Phenix were carried out.

Water and the appropriate ligands were added manually by examination of the Fo-Fc and 2Fo-Fc electron density maps. In all structures, all ligands were refined at full occupancy. Figures 5. Acylation and deacylation experiments were performed as described previously , For all compounds described, on-rates were determined using a continuous assay with nitrocefin as reporter substrate.

The maximum concentration of inhibitor used for CTX-M on-rates was: avibactam, 0. The same methods were applied for OXA 0. For concentration-response experiments assay buffer was used as above. After 30 minutes of treatment, cells were disrupted by a French press and the bacterial lysate was centrifuged at 12, g for 10 min to remove unbroken cells. Protein concentration was estimated by the method of Bradford with the BCA kit Pierce using bovine serum albumin as a standard. All reagents were diluted in assay buffer prior to use. To start the reaction, various concentrations of unlabeled compound and In contrast, for pre-incubation experiments various amounts of competitor compound were pre-incubated with 4.

Densitometry analysis was performed using ImageJ as previously described The individual data points were normalized to the maximum value of the fluorescence intensity, which represents total saturation of protein by BOCILLIN FL in the absence of unlabeled compound. Benzyl penicillin, and Kanamycin were used as positive and negative controls, respectively.