Contents 1. Introduction 395 2. Penicillin-Binding Proteins 398 2.1. Enzymes of Cell Wall Biosynthesis 398 2.2. β-Lactam-Sensing Proteins 402 3. β-Lactamases 404 3.1. Overview and Classification 404 3.2. Class A β-Lactamases 406 3.3. Class C β-Lactamases 411 3.4. Class D β-Lactamases 412 3.5. Class B Metallo-β-lactamases 413 4. Other Resistance Mechanisms 416 4.1. Porin Deletion 416 4.2. Transporter Expression 417 5. Envoi 417 6. Acknowledgments 419 7. Abbreviations 419 8. Note Added in Proof 419 9. References 419 Scheme 1

The 3D structure of the bacterial peptidoglycan, the major constituent of the cell wall, is one of the most important, yet still unsolved, structural problems in biochemistry. The peptidoglycan comprises alternating N-acetylglucosamine (NAG) and N-acetylmuramic disaccharide (NAM) saccharides, the latter of which has a peptide stem. Adjacent peptide stems are cross-linked by the transpeptidase enzymes of cell wall biosynthesis to provide the cell wall polymer with the structural integrity required by the bacterium. The cell wall and its biosynthetic enzymes are targets of antibiotics. The 3D structure of the cell wall has been elusive because of its complexity and the lack of pure samples. Herein we report the 3D solution structure as determined by NMR of the 2-kDa NAG-NAM(pentapeptide)-NAG-NAM(pentapeptide) synthetic fragment of the cell wall. The glycan backbone of this peptidoglycan forms a right-handed helix with a periodicity of three for the NAG-NAM repeat (per turn of the helix). The first two amino acids of the pentapeptide adopt a limited number of conformations. Based on this structure a model for the bacterial cell wall is proposed. This pentapeptide stem participates in an interglycan cross-linking reaction, thus creating the cell wall polymer. In contrast to the two other ␤-1,4-linked glycan biopolymers, cellulose (repeating glucose) (1-4) and chitin (repeating NAG) (5-7) for which the 3D structure is solved, the structure of the bacterial cell wall has remained elusive because of its complexity and the lack of pure and discrete segments for structural study (8-18). Herein we describe the 3D structure, determined in aqueous solution by NMR, of a 2-kDa synthetic NAG-NAM(pentapeptide)-NAG-NAM(pentapeptide) tetrasaccharide cell wall segment. The defining aspect of this structure is an ordered, right-handed helical saccharide conformation corresponding to three NAG-NAM pairs per turn of the helix. The structure of this peptidoglycan segment is the basis for a proposal for the structure of the bacterial cell wall polymer.Results and Discussion 3D Structure of the Peptidoglycan. Because of the critical significance of the cell wall to bacterial survival, and the exploitation of the cell wall biosynthetic enzymes for the chemotherapeutic intervention of infections, many experimental and theoretical studies have addressed the cell wall structure. Despite diffraction studies carried out Ͼ30 years ago on cell wall extracted from bacteria, which strongly suggested that the peptidoglycan polymer possessed regular order (11), the 3D structure of the cell wall is not known. An excellent account of the historical development of the hypotheses for the cell wall structure is given by Dmitriev, Toukach, and Ehlers in their recent review (18). The major reason for the lack of progress is the absence of a pure fragment of the cell wall, having both the peptide and disaccharide components of the peptidoglycan, for structural investigation. To address this limitation we completed the 37-step synthesis of such a segment (1...

For Abstract see ChemInform Abstract in Full Text.

β-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 β-lactamases exist, attempts to correlate structure and function have become daunting. Although new enzymes in the extended-spectrum β-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-β-lactamases VIM, IMP, and NDM-1, even though NDM-1 was only recently identified. The rapid emergence of these newer enzymes, with multiple β-lactamases appearing in a single organism, makes the design of new β-lactamase inactivators or β-lactamase-stable β-lactams all the more difficult. Combination therapy will likely be required to counteract the continuing evolution of these insidious enzymes in multidrug-resistant pathogens.

Many Gram-negative and Gram-positive bacteria recycle a significant proportion of the peptidoglycan components of their cell walls during their growth and septation. In many—and quite possibly all—bacteria, the peptidoglycan fragments are recovered and recycled. While cell-wall recycling is beneficial for the recovery of resources, it also serves as a mechanism to detect cell-wall–targeting antibiotics and to regulate resistance mechanisms. In several Gram-negative pathogens, anhydro-MurNAc-peptide cell-wall fragments regulate AmpC β-lactamase induction. In some Gram-positive organisms, short peptides derived from the cell wall regulate the induction of both β-lactamase and β-lactam-resistant penicillin-binding proteins. The involvement of peptidoglycan recycling with resistance regulation suggests that inhibitors of the enzymes involved in the recycling might synergize with cell-wall-targeted antibiotics. Indeed, such inhibitors improve the potency of β-lactams in vitro against inducible AmpC β-lactamase-producing bacteria. We describe the key steps of cell-wall remodeling and recycling, the regulation of resistance mechanisms by cell-wall recycling, and recent advances toward the discovery of cell-wall recycling inhibitors.

The expression of penicillin binding protein 2a (PBP2a) is the basis for the broad clinical resistance to the β-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 β-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 β-lactam antibiotics. In contrast, the PBP2a of MRSA is resistant to β-lactam acylation and successfully catalyzes the DD-transpeptidation reaction necessary to complete the cell wall. The inability to contain MRSA infection with β-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 β-lactam antibiotic. The ability of an anti-MRSA β-lactam antibiotic to stimulate allosteric opening of the active site, thus predisposing PBP2a to inactivation by a second β-lactam molecule, opens an unprecedented realm for β-lactam antibiotic structure-based design.

Beta-lactamase acquisition is the most prevalent basis for Gram-negative bacteria resistance to the beta-lactam antibiotics. The mechanism used by the most common class A Gram-negative beta-lactamases is serine acylation followed by hydrolytic deacylation, destroying the beta-lactam. The ab initio quantum mechanical/molecular mechanical (QM/MM) calculations, augmented by extensive molecular dynamics simulations reported herein, describe the serine acylation mechanism for the class A TEM-1 beta-lactamase with penicillanic acid as substrate. Potential energy surfaces (based on approximately 350 MP2/6-31+G calculations) reveal the proton movements that govern Ser70 tetrahedral formation and then collapse to the acyl-enzyme. A remarkable duality of mechanism for tetrahedral formation is implicated. Following substrate binding, the pathway initiates by a low energy barrier (5 kcal mol(-1)) and an energetically favorable transfer of a proton from Lys73 to Glu166, through the catalytic water molecule and Ser70. This gives unprotonated Lys73 and protonated Glu166. Tetrahedral formation ensues in a concerted general base process, with Lys73 promoting Ser70 addition to the beta-lactam carbonyl. Moreover, the three-dimensional potential energy surface also shows that the previously proposed pathway, involving Glu166 as the general base promoting Ser70 through a conserved water molecule, exists in competition with the Lys73 process. The existence of two routes to the tetrahedral species is fully consistent with experimental data for mutant variants of the TEM beta-lactamase.

The search for an MMP inhibitor with anticancer efficacy is a nearly three-decade endeavor. This inhibitor is yet to be found. The reasons for this failure include shortcomings in the chemistry of these compounds (including broad MMP sub-type selectivity, metabolic lability, and toxicity) as well as the emerging, and arguably extraordinary, complexity of MMP cell (and cancer) biology. Together these suggest that the successful anticancer inhibitor must possess MMP selectivity against the MMP subtype whose involvement is critical, yet highly temporally (with respect to metastatic progression) and mechanistically (with respect to matrix degradation) regulated. This review summarizes the progression of chemical structure and mechanistic thinking toward these objectives, with emphasis on the disappointment, the perseverance, and the resilient optimism that such an inhibitor is there to be discovered.