Catalytic process of anhydro-N-acetylmuramic acid kinase from Pseudomonas aeruginosa.

The bacterial cell envelope is the structure with which the bacterium engages with, and is protected from, its environment. Within this envelop is a conserved peptidoglycan polymer which confers shape and strength to the cell envelop. The enzymatic processes that build, remodel, and recycle the chemical components of this cross-linked polymer are preeminent targets of antibiotics and exploratory targets for emerging antibiotic structures. We report a comprehensive kinetic and structural analysis for one such enzyme, the Pseudomonas aeruginosa anhydro-N-acetylmuramic acid (anhNAM) kinase (AnmK). AnmK is an enzyme in the peptidoglycan-recycling pathway of this pathogen. It catalyzes the pairing of hydrolytic ring opening of anhNAM with concomitant ATP-dependent phosphoryl transfer. AnmK follows a random-sequential kinetic mechanism with respect to its anhNAM and ATP substrates. Crystallographic analyses of four distinct structures (apo AnmK, AnmK:AMPPNP, AnmK:AMPPNP:anhNAM, and AnmK:ATP:anhNAM) demonstrate that both substrates enter the active site independently in an ungated conformation of the substrate subsites, with protein loops acting as gates for anhNAM binding. Catalysis occurs within a closed conformational state for the enzyme. We observe this state crystallographically using ATP-mimetic molecules. A remarkable X-ray structure for dimeric AnmK sheds light on the precatalytic and postcatalytic ternary complexes. Computational simulations in conjunction with the high-resolution X-ray structures reveal the full catalytic cycle. We further report that a P. aeruginosa strain with disrupted anmK gene is more susceptible to the ß-lactam imipenem compared to the WT strain. These observations position AnmK for understanding the nexus among peptidoglycan recycling, susceptibility to antibiotics, and bacterial virulence.

ß-Lactams against the Fortress of the Gram-Positive Staphylococcus aureus Bacterium.

The biological diversity of the unicellular bacteria-whether assessed by shape, food, metabolism, or ecological niche-surely rivals (if not exceeds) that of the multicellular eukaryotes. The relationship between bacteria whose ecological niche is the eukaryote, and the eukaryote, is often symbiosis or stasis. Some bacteria, however, seek advantage in this relationship. One of the most successful-to the disadvantage of the eukaryote-is the small (less than 1 µm diameter) and nearly spherical Staphylococcus aureus bacterium. For decades, successful clinical control of its infection has been accomplished using ß-lactam antibiotics such as the penicillins and the cephalosporins. Over these same decades S. aureus has perfected resistance mechanisms against these antibiotics, which are then countered by new generations of ß-lactam structure. This review addresses the current breadth of biochemical and microbiological efforts to preserve the future of the ß-lactam antibiotics through a better understanding of how S. aureus protects the enzyme targets of the ß-lactams, the penicillin-binding proteins. The penicillin-binding proteins are essential enzyme catalysts for the biosynthesis of the cell wall, and understanding how this cell wall is integrated into the protective cell envelope of the bacterium may identify new antibacterials and new adjuvants that preserve the efficacy of the ß-lactams.

ß-Lactams from the Ocean.

The title of this essay is as much a question as it is a statement. The discovery of the ß-lactam antibiotics-including penicillins, cephalosporins, and carbapenems-as largely (if not exclusively) secondary metabolites of terrestrial fungi and bacteria, transformed modern medicine. The antibiotic ß-lactams inactivate essential enzymes of bacterial cell-wall biosynthesis. Moreover, the ability of the ß-lactams to function as enzyme inhibitors is of such great medical value, that inhibitors of the enzymes which degrade hydrolytically the ß-lactams, the ß-lactamases, have equal value. Given this privileged status for the ß-lactam ring, it is therefore a disappointment that the exemplification of this ring in marine secondary metabolites is sparse. It may be that biologically active marine ß-lactams are there, and simply have yet to be encountered. In this report, we posit a second explanation: that the value of the ß-lactam to secure an ecological advantage in the marine environment might be compromised by its close structural similarity to the ß-lactones of quorum sensing. The steric and reactivity similarities between the ß-lactams and the ß-lactones represent an outside-of-the-box opportunity for correlating new structures and new enzyme targets for the discovery of compelling biological activities.

Cell-Wall Recycling of the Gram-Negative Bacteria and the Nexus to Antibiotic Resistance.

The importance of the cell wall to the viability of the bacterium is underscored by the breadth of antibiotic structures that act by blocking key enzymes that are tasked with cell-wall creation, preservation, and regulation. The interplay between cell-wall integrity, and the summoning forth of resistance mechanisms to deactivate cell-wall-targeting antibiotics, involves exquisite orchestration among cell-wall synthesis and remodeling and the detection of and response to the antibiotics through modulation of gene regulation by specific effectors. Given the profound importance of antibiotics to the practice of medicine, the assertion that understanding this interplay is among the most fundamentally important questions in bacterial physiology is credible. The enigmatic regulation of the expression of the AmpC ß-lactamase, a clinically significant and highly regulated resistance response of certain Gram-negative bacteria to the ß-lactam antibiotics, is the exemplar of this challenge. This review gives a current perspective to this compelling, and still not fully solved, 35-year enigma.

Lytic transglycosylases: concinnity in concision of the bacterial cell wall.

The lytic transglycosylases (LTs) are bacterial enzymes that catalyze the non-hydrolytic cleavage of the peptidoglycan structures of the bacterial cell wall. They are not catalysts of glycan synthesis as might be surmised from their name. Notwithstanding the seemingly mundane reaction catalyzed by the LTs, their lytic reactions serve bacteria for a series of astonishingly diverse purposes. These purposes include cell-wall synthesis, remodeling, and degradation; for the detection of cell-wall-acting antibiotics; for the expression of the mechanism of cell-wall-acting antibiotics; for the insertion of secretion systems and flagellar assemblies into the cell wall; as a virulence mechanism during infection by certain Gram-negative bacteria; and in the sporulation and germination of Gram-positive spores. Significant advances in the mechanistic understanding of each of these processes have coincided with the successive discovery of new LTs structures. In this review, we provide a systematic perspective on what is known on the structure-function correlations for the LTs, while simultaneously identifying numerous opportunities for the future study of these enigmatic enzymes.

A Structural Dissection of the Active Site of the Lytic Transglycosylase MltE from Escherichia coli.

Lytic transglycosylases (LTs) are bacterial enzymes that catalyze the cleavage of the glycan strands of the bacterial cell wall. The mechanism of this cleavage is a remarkable intramolecular transacetalization reaction, accomplished by an ensemble of active-site residues. Because the LT reaction occurs in parallel with the cell wall bond-forming reactions catalyzed by the penicillin-binding proteins, simultaneous inhibition of both enzymes can be particularly bactericidal to Gram-negative bacteria. The MltE lytic transglycosylase is the smallest of the eight LTs encoded by the Escherichia coli genome. Prior crystallographic and computational studies identified four active-site residues-E64, S73, S75, and Y192-as playing roles in catalysis. Each of these four residues was individually altered by mutation to give four variant enzymes (E64Q, S73A, S75A, and Y192F). All four variants showed reduced catalytic activity [soluble wild type (100%) > soluble Y192F and S75A (both 40%) > S73A (4%) > E64Q (≤1%)]. The crystal structure of each variant protein was determined at the resolution of 2.12 Å for E64Q, 2.33 Å for Y192F, 1.38 Å for S73A, and 1.35 Å for S75A. These variants show alteration of the hydrogen-bond interactions of the active site. Within the framework of a prior computational study of the LT mechanism, we suggest the mechanistic role of these four active-site residues in MltE catalysis.

Conformational Dynamics in Penicillin-Binding Protein 2a of Methicillin-Resistant Staphylococcus aureus, Allosteric Communication Network and Enablement of Catalysis.

The mechanism of the ß-lactam antibacterials is the functionally irreversible acylation of the enzymes that catalyze the cross-linking steps in the biosynthesis of their peptidoglycan cell wall. The Gram-positive pathogen Staphylococcus aureus uses one primary resistance mechanism. An enzyme, called penicillin-binding protein 2a (PBP2a), is brought into this biosynthetic pathway to complete the cross-linking. PBP2a effectively discriminates against the ß-lactam antibiotics as potential inhibitors, and in favor of the peptidoglycan substrate. The basis for this discrimination is an allosteric site, distal from the active site, that when properly occupied concomitantly opens the gatekeeper residues within the active site and realigns the conformation of key residues to permit catalysis. We address the molecular basis of this regulation using crystallographic studies augmented by computational analyses. The crystal structures of three ß-lactams (oxacillin, cefepime, ceftazidime) complexes with PBP2a-each with the ß-lactam in the allosteric site-defined (with preceding PBP2a structures) as the "open" or "partially open" PBP2a states. A particular loop motion adjacent to the active site is identified as the driving force for the active-site conformational change that accompanies active-site opening. Correlation of this loop motion to effector binding at the allosteric site, in order to identify the signaling pathway, was accomplished computationally in reference to the known "closed" apo-PBP2a X-ray crystal structure state. This correlation enabled the computational simulation of the structures coinciding with initial peptidoglycan substrate binding to PBP2a, acyl enzyme formation, and acyl transfer to a second peptidoglycan substrate to attain cross-linking. These studies offer important insights into the structural bases for allosteric site-to-active site communication and for ß-lactam mimicry of the peptidoglycan substrates, as foundational to the mechanistic understanding of emerging PBP2a resistance mutations.

Muropeptide Binding and the X-ray Structure of the Effector Domain of the Transcriptional Regulator AmpR of Pseudomonas aeruginosa.

A complex link exists between cell-wall recycling/repair and the manifestation of resistance to ß-lactam antibiotics in many Enterobacteriaceae and Pseudomonas aeruginosa. This process is mediated by specific cell-wall-derived muropeptide products. These muropeptides are internalized into the cytoplasm and bind to the transcriptional regulator AmpR, which controls the cytoplasmic events that lead to expression of ß-lactamase, an antibiotic-resistance determinant. The effector-binding domain (EBD) of AmpR was purified to homogeneity. We document that the EBD exists exclusively as a dimer, even at a concentration as low as 1 µM. The EBD binds to the suppressor ligand UDP-N-acetyl-ß-d-muramyl-l-Ala-γ-d-Glu-meso-DAP-d-Ala-d-Ala and binds to two activator muropeptides, N-acetyl-ß-d-glucosamine-(1→4)-1,6-anhydro-N-acetyl-ß-d-muramyl-l-Ala-γ-d-Glu-meso-DAP-d-Ala-d-Ala and 1,6-anhydro-N-acetyl-ß-d-muramyl-l-Ala-γ-d-Glu-meso-DAP-d-Ala-d-Ala, as assessed by non-denaturing mass spectrometry. The EBD does not bind to 1,6-anhydro-N-acetyl-ß-d-muramyl-l-Ala-γ-d-Glu-meso-DAP. This binding selectivity revises the dogma in the field. The crystal structure of the EBD dimer was solved to 2.2 Å resolution. The EBD crystallizes in a "closed" conformation, in contrast to the "open" structure required to bind the muropeptides. Structural issues of this ligand recognition are addressed by molecular dynamics simulations, which reveal significant differences among the complexes with the effector molecules.

From Genome to Proteome to Elucidation of Reactions for All Eleven Known Lytic Transglycosylases from Pseudomonas aeruginosa.

An enzyme superfamily, the lytic transglycosylases (LTs), occupies the space between the two membranes of Gram-negative bacteria. LTs catalyze the non-hydrolytic cleavage of the bacterial peptidoglycan cell-wall polymer. This reaction is central to the growth of the cell wall, for excavating the cell wall for protein insertion, and for monitoring the cell wall so as to initiate resistance responses to cell-wall-acting antibiotics. The nefarious Gram-negative pathogen Pseudomonas aeruginosa encodes eleven LTs. With few exceptions, their substrates and functions are unknown. Each P. aeruginosa LT was expressed as a soluble protein and evaluated with a panel of substrates (both simple and complex mimetics of their natural substrates). Thirty-one distinct products distinguish these LTs with respect to substrate recognition, catalytic activity, and relative exolytic or endolytic ability. These properties are foundational to an understanding of the LTs as catalysts and as antibiotic targets.

Epidemiological expansion, structural studies, and clinical challenges of new ß-lactamases from gram-negative bacteria.

ß-Lactamase evolution presents to the infectious disease community a major challenge in the treatment of infections caused by multidrug-resistant gram-negative bacteria. Because over 1,000 of these naturally occurring ß-lactamases exist, attempts to correlate structure and function have become daunting. Although new enzymes in the extended-spectrum ß-lactamase (ESBL) families are frequently identified, the older CTX-M-14 and CTX-M-15 enzymes have become the most prevalent ESBLs in global surveillance. Carbapenemases with either serine-based or zinc-facilitated hydrolysis mechanisms are posing some of the most critical problems. Most geographical regions now report KPC serine carbapenemases and the metallo-ß-lactamases VIM, IMP, and NDM-1, even though NDM-1 was only recently identified. The rapid emergence of these newer enzymes, with multiple ß-lactamases appearing in a single organism, makes the design of new ß-lactamase inactivators or ß-lactamase-stable ß-lactams all the more difficult. Combination therapy will likely be required to counteract the continuing evolution of these insidious enzymes in multidrug-resistant pathogens.

Ensemble of Pinanones from the Permanganate Oxidation of Myrtenal.

The buffered permanganate oxidation of (-)-myternal, a member of the pinene family, provides the α-hydroxyketone (-)-(1R,3S,5R)-3-hydroxy-6,6-dimethylbicyclo[3.1.1]heptan-2-one in preparative yield (65% on a multigram scale). This α-hydroxyketone is oxidized, in a second reaction, to the α,ß-diketone (1R,5R)-6,6-dimethylbicyclo[3.1.1]heptane-2,3-dione ("PinDione"). As both oxidations are fast, simple, safe, inexpensive, good-yielding, and multigram scalable, these transformations are a preparative expansion of the pinane family.

Three-dimensional QSAR analysis and design of new 1,2,4-oxadiazole antibacterials.

The oxadiazole antibacterials, a class of newly discovered compounds that are active against Gram-positive bacteria, target bacterial cell-wall biosynthesis by inhibition of a family of essential enzymes, the penicillin-binding proteins. Ligand-based 3D-QSAR analyses by comparative molecular field analysis (CoMFA), comparative molecular shape indices analysis (CoMSIA) and Field-Based 3D-QSAR evaluated a series of 102 members of this class. This series included inactive compounds as well as compounds that were moderately to strongly antibacterial against Staphylococcus aureus. Multiple models were constructed using different types of energy minimization and charge calculations. CoMFA derived contour maps successfully defined favored and disfavored regions of the molecules in terms of steric and electrostatic properties for substitution.

How allosteric control of Staphylococcus aureus penicillin binding protein 2a enables methicillin resistance and physiological function.

The expression of penicillin binding protein 2a (PBP2a) is the basis for the broad clinical resistance to the ß-lactam antibiotics by methicillin-resistant Staphylococcus aureus (MRSA). The high-molecular mass penicillin binding proteins of bacteria catalyze in separate domains the transglycosylase and transpeptidase activities required for the biosynthesis of the peptidoglycan polymer that comprises the bacterial cell wall. In bacteria susceptible to ß-lactam antibiotics, the transpeptidase activity of their penicillin binding proteins (PBPs) is lost as a result of irreversible acylation of an active site serine by the ß-lactam antibiotics. In contrast, the PBP2a of MRSA is resistant to ß-lactam acylation and successfully catalyzes the DD-transpeptidation reaction necessary to complete the cell wall. The inability to contain MRSA infection with ß-lactam antibiotics is a continuing public health concern. We report herein the identification of an allosteric binding domain--a remarkable 60 Å distant from the DD-transpeptidase active site--discovered by crystallographic analysis of a soluble construct of PBP2a. When this allosteric site is occupied, a multiresidue conformational change culminates in the opening of the active site to permit substrate entry. This same crystallographic analysis also reveals the identity of three allosteric ligands: muramic acid (a saccharide component of the peptidoglycan), the cell wall peptidoglycan, and ceftaroline, a recently approved anti-MRSA ß-lactam antibiotic. The ability of an anti-MRSA ß-lactam antibiotic to stimulate allosteric opening of the active site, thus predisposing PBP2a to inactivation by a second ß-lactam molecule, opens an unprecedented realm for ß-lactam antibiotic structure-based design.

The Tipper-Strominger Hypothesis and Triggering of Allostery in Penicillin-Binding Protein 2a of Methicillin-Resistant Staphylococcus aureus (MRSA).

The transpeptidases involved in the synthesis of the bacterial cell wall (also known as penicillin-binding proteins, PBPs) have evolved to bind the acyl-D-Ala-D-Ala segment of the stem peptide of the nascent peptidoglycan for the physiologically important cross-linking of the cell wall. The Tipper-Strominger hypothesis stipulates that ß-lactam antibiotics mimic the acyl-D-Ala-D-Ala moiety of the stem and, thus, are recognized by the PBPs with bactericidal consequences. We document that this mimicry exists also at the allosteric site of PBP2a of methicillin-resistant Staphylococcus aureus (MRSA). Interactions of different classes of ß-lactam antibiotics, as mimics of the acyl-D-Ala-D-Ala moiety at the allosteric site, lead to a conformational change, across a distance of 60 Å to the active site. We directly visualize this change using an environmentally sensitive fluorescent probe affixed to the protein loops that frame the active site. This conformational mobility, documented in real time, allows antibiotic access to the active site of PBP2a. Furthermore, we document that this allosteric trigger enables synergy between two different ß-lactam antibiotics, wherein occupancy at the allosteric site by one facilitates occupancy by a second at the transpeptidase catalytic site, thus lowering the minimal-inhibitory concentration. This synergy has important implications for the mitigation of facile emergence of resistance to these antibiotics by MRSA.

Catalytic spectrum of the penicillin-binding protein 4 of Pseudomonas aeruginosa, a nexus for the induction of ß-lactam antibiotic resistance.

Pseudomonas aeruginosa is an opportunistic Gram-negative bacterial pathogen. A primary contributor to its ability to resist ß-lactam antibiotics is the expression, following detection of the ß-lactam, of the AmpC ß-lactamase. As AmpC expression is directly linked to the recycling of the peptidoglycan of the bacterial cell wall, an important question is the identity of the signaling molecule(s) in this relationship. One mechanism used by clinical strains to elevate AmpC expression is loss of function of penicillin-binding protein 4 (PBP4). As the mechanism of the ß-lactams is PBP inactivation, this result implies that the loss of the catalytic function of PBP4 ultimately leads to induction of antibiotic resistance. PBP4 is a bifunctional enzyme having both dd-carboxypeptidase and endopeptidase activities. Substrates for both the dd-carboxypeptidase and the 4,3-endopeptidase activities were prepared by multistep synthesis, and their turnover competence with respect to PBP4 was evaluated. The endopeptidase activity is specific to hydrolysis of 4,3-cross-linked peptidoglycan. PBP4 catalyzes both reactions equally well. When P. aeruginosa is grown in the presence of a strong inducer of AmpC, the quantities of both the stem pentapeptide (the substrate for the dd-carboxypeptidase activity) and the 4,3-cross-linked peptidoglycan (the substrate for the 4,3-endopeptidase activity) increase. In the presence of ß-lactam antibiotics these altered cell-wall segments enter into the muropeptide recycling pathway, the conduit connecting the sensing event in the periplasm and the unleashing of resistance mechanisms in the cytoplasm.

Regioselective Control of the SNAr Amination of 5-Substituted-2,4-Dichloropyrimidines Using Tertiary Amine Nucleophiles.

The SNAr reaction of 2,4-dichloropyrimidines, further substituted with an electron-withdrawing substituent at C-5, has selectivity for substitution at C-4. Here we report that tertiary amine nucleophiles show excellent C-2 selectivity. In situ N-dealkylation of an intermediate gives the product that formally corresponds to the reaction of a secondary amine nucleophile at C-2. This reaction is practical (fast under simple reaction conditions, with good generality for tertiary amine structure and moderate to excellent yields) and significantly expands access to pyrimidine structures.

Protonation states of active-site lysines of penicillin-binding protein 6 from Escherichia coli and the mechanistic implications.

The protonation states of the two active-site lysines (Lys69 and Lys235) of PBP 6 of Escherichia coli were explored to understand the active site chemistry of this enzyme. Each lysine was individually mutated to cysteine, and the resultant two mutant proteins were purified to homogeneity. Each protein was denatured, and its cysteine was chemically modified to produce an S-aminoethylated cysteine (γ-thialysine) residue. Following renaturation, the evaluation of the kinetics of the dd-carboxypeptidase activity of PBP 6 as a function of pH was found consistent with one lysine in its free-base (Lys69) and the other in the protonated state (Lys235) for optimal catalysis. The experimental estimates for their pKa values were compared with the pKa values calculated computationally, using molecular-dynamics simulations and a thermodynamic cycle. Study of the γ-thialysine69 showed that lysine at position 69 influenced the basic limb of catalysis, consistent with the fact that the two lysine side chains are in proximity to each other in the active site. Based on these observations, a reaction sequence for PBP 6 is proposed, wherein protonated Lys235 serves as the electrostatic substrate anchor and Lys69 as the conduit for protons in the course of the acylation and deacylation half-reactions.

The sentinel role of peptidoglycan recycling in the ß-lactam resistance of the Gram-negative Enterobacteriaceae and Pseudomonas aeruginosa.

The peptidoglycan is the structural polymer of the bacterial cell envelope. In contrast to an expectation of a structural stasis for this polymer, during the growth of the Gram-negative bacterium this polymer is in a constant state of remodeling and extension. Our current understanding of this peptidoglycan "turnover" intertwines with the deeply related phenomena of the liberation of small peptidoglycan segments (muropeptides) during turnover, the presence of dedicated recycling pathways for reuse of these muropeptides, ß-lactam inactivation of specific penicillin-binding proteins as a mechanism for the perturbation of the muropeptide pool, and this perturbation as a controlling mechanism for signal transduction leading to the expression of ß-lactamase(s) as a key resistance mechanism against the ß-lactam antibiotics. The nexus for many of these events is the control of the AmpR transcription factor by the composition of the muropeptide pool generated during peptidoglycan recycling. In this review we connect the seminal observations of the past decades to new observations that resolve some, but certainly not all, of the key structures and mechanisms that connect to AmpR.

Lytic transglycosylase Slt of Pseudomonas aeruginosa as a periplasmic hub protein.

Peptidoglycan is a major constituent of the bacterial cell wall. Its integrity as a polymeric edifice is critical for bacterial survival and, as such, it is a preeminent target for antibiotics. The peptidoglycan is a dynamic crosslinked polymer that undergoes constant biosynthesis and turnover. The soluble lytic transglycosylase (Slt) of Pseudomonas aeruginosa is a periplasmic enzyme involved in this dynamic turnover. Using amber-codon-suppression methodology in live bacteria, we incorporated a fluorescent chromophore into the structure of Slt. Fluorescent microscopy shows that Slt populates the length of the periplasmic space and concentrates at the sites of septation in daughter cells. This concentration persists after separation of the cells. Amber-codon-suppression methodology was also used to incorporate a photoaffinity amino acid for the capture of partner proteins. Mass-spectrometry-based proteomics identified 12 partners for Slt in vivo. These proteomics experiments were complemented with in vitro pulldown analyses. Twenty additional partners were identified. We cloned the genes and purified to homogeneity 22 identified partners. Biophysical characterization confirmed all as bona fide Slt binders. The identities of the protein partners of Slt span disparate periplasmic protein families, inclusive of several proteins known to be present in the divisome. Notable periplasmic partners (KD < 0.5 µM) include PBPs (PBP1a, KD = 0.07 µM; PBP5 = 0.4 µM); other lytic transglycosylases (SltB2, KD = 0.09 µM; RlpA, KD = 0.4 µM); a type VI secretion system effector (Tse5, KD = 0.3 µM); and a regulatory protease for alginate biosynthesis (AlgO, KD < 0.4 µM). In light of the functional breadth of its interactome, Slt is conceptualized as a hub protein within the periplasm.

Structural analysis of the role of Pseudomonas aeruginosa penicillin-binding protein 5 in ß-lactam resistance.

Penicillin-binding protein 5 (PBP5) is one of the most abundant PBPs in Pseudomonas aeruginosa. Although its main function is that of a cell wall dd-carboxypeptidase, it possesses sufficient ß-lactamase activity to contribute to the ability of P. aeruginosa to resist the antibiotic activity of the ß-lactams. The study of these dual activities is important for understanding the mechanisms of antibiotic resistance by P. aeruginosa, an important human pathogen, and to the understanding of the evolution of ß-lactamase activity from the PBP enzymes. We purified a soluble version of P. aeruginosa PBP5 (designated Pa sPBP5) by deletion of its C-terminal membrane anchor. Under in vitro conditions, Pa sPBP5 demonstrates both dd-carboxypeptidase and expanded-spectrum ß-lactamase activities. Its crystal structure at a 2.05-Å resolution shows features closely resembling those of the class A ß-lactamases, including a shortened loop spanning residues 74 to 78 near the active site and with respect to the conformations adopted by two active-site residues, Ser101 and Lys203. These features are absent in the related PBP5 of Escherichia coli. A comparison of the two Pa sPBP5 monomers in the asymmetric unit, together with molecular dynamics simulations, revealed an active-site flexibility that may explain its carbapenemase activity, a function that is absent in the E. coli PBP5 enzyme. Our functional and structural characterizations underscore the versatility of this PBP5 in contributing to the ß-lactam resistance of P. aeruginosa while highlighting how broader ß-lactamase activity may be encoded in the structural folds shared by the PBP and serine ß-lactamase classes.