Beta-lactam resistance in Staphylococcus aureus: the adaptive resistance of a plastic genome.

Staphylococci have two mechanisms for resistance to beta-lactam antibiotics. One is the production of beta-lactamases, enzymes that hydrolytically destroy beta-lactams. The other is the expression of penicillin-binding protein 2a (PBP 2a), which is not susceptible to inhibition by beta-lactam antibiotics. Strains of S. aureus exhibiting either beta-lactamase or PBP 2a-directed resistance (or both) have established a considerable ecological niche among human pathogens. The emergence and subsequent spread of bacterial strains designated as methicillin-resistant S. aureus (MRSA), from the 1960s to the present, has created clinical difficulties for nosocomial treatment on a global scale. The recent variants of MRSA that are resistant to glycopeptide antibiotics (such as vancomycin) have ushered in a new and disconcerting chapter in the evolution of this organism.

Conjoint molecules of cephalosporins and aminoglycosides.

A general synthetic route to conjoint molecules of cephalosporins and aminoglycosides is described. These molecules were designed as potential substrates for bacterial beta-lactamases, enzymes that hydrolyze the beta-lactam bond of cephalosporins. Hydrolysis of the beta-lactam bond was expected to release the C10-appended aminoglycoside. Since beta-lactamases are sequestered in the periplasmic space of gram-negative bacteria, this sequence of events would liberate aminoglycoside inside such bacteria. It is expected that such local delivery of aminoglycosides would circumvent the inherent toxicity of aminoglycosides that occurs during systemic exposure within the mammalian host.

Critical involvement of a carbamylated lysine in catalytic function of class D beta-lactamases.

beta-Lactamases are the resistance enzymes for beta-lactam antibiotics, of which four classes are known. beta-lactamases hydrolyze the beta-lactam moieties of these antibiotics, rendering them inactive. It is shown herein that the class D OXA-10 beta-lactamase depends critically on an unusual carbamylated lysine as the basic residue for both the enzyme acylation and deacylation steps of catalysis. The formation of carbamylated lysine is reversible. Evidence is presented that this enzyme is dimeric and carbamylated in living bacteria. High-resolution x-ray structures for the native enzyme were determined at pH values of 6.0, 6.5, 7.5, and 8.5. Two dimers are present per asymmetric unit. One monomer in each dimer was carbamylated at pH 6.0, whereas all four monomers were fully carbamylated at pH 8.5. At the intermediate pH values, one monomer of each dimer was carbamylated, and the other showed a mixture of carbamylated and non-carbamylated lysines. It would appear that, as the pH increased for the sample, additional lysines were "titrated" by carbamylation. A handful of carbamylated lysines are known from protein crystallographic data, all of which have been attributed roles in structural stabilization (mostly as metal ligands) of the proteins. This paper reports a previously unrecognized role for a noncoordinated carbamylate lysine as a basic residue involved in mechanistic reactions of an enzyme, which indicates another means for expansion of the catalytic capabilities of the amino acids in nature beyond the 20 common amino acids in development of biological catalysts.

Identification of colchicine in placental blood from patients using herbal medicines.

While characterizing natural antiinflammatory substances in human placental blood, we discovered a factor that affected human neutrophils and their adherence. Rigorous chemical and stereochemical analyses revealed this factor to be the well-known alkaloid, colchicine. When samples from individual patients were analyzed, significant levels (49-763 microg/L) of colchicine could be found in placental blood of patients using nonprescription herbal dietary supplements during pregnancy. We confirmed the presence of colchicine in commercially available ginkgo biloba. Due to its potential harmful effects, it would appear that such supplements should be avoided by women who are pregnant or are trying to conceive.

Aminoglycoside-modifying enzymes: mechanisms of catalytic processes and inhibition.

The most prevalent mechanism for resistance to aminoglycoside antibiotics is mediated through their enzymatic modification in resistant organisms. Dozens of aminoglycoside-modifying enzymes are known at the gene sequence level, but few have been characterized in the details of their mechanism. This review summarizes the state of knowledge of the best studied of these enzymes, focusing on their catalytic mechanisms and inhibition.

Inhibition of beta-lactamases by 6,6-bis(hydroxylmethyl)penicillanate.

beta-Lactamases of classes A and C are the two most prevalent resistant determinants to beta-lactam antibiotics among bacterial pathogens. Both these enzymes pursue different mechanisms for their catalytic processes, highlighted by the fact that the hydrolytic water molecule in each approaches the ester of the intermediary acyl-enzyme species from the opposite ends. 6,6-Bis(hydroxylmethyl)penicillanate was designed as an inhibitor that would impair the approach of the hydrolytic water molecule in either of these enzymes upon formation of the acyl-enzyme species. The design, synthesis, and kinetic evaluation of this inhibitor are disclosed herein.

Insight into the complex and dynamic process of activation of matrix metalloproteinases.

Matrix metalloproteinases (MMPs) are important hydrolytic enzymes with profound physiological and pathological functions in living organisms. MMPs are produced in their inactive zymogenic forms, which are subsequently proteolytically activated in an elaborate set of events. The propeptide in the zymogen blocks the active site, with a cysteine side-chain thiolate from this propeptide achieving coordination with the catalytically important zinc ion in the active site. Molecular dynamics simulations, ab initio calculations, and wet chemistry experiments presented herein argue for the critical importance of a protonation event at the coordinated thiolate as a prerequisite for the departure of the propeptide from the active site. Furthermore, a catalytically important glutamate is shown to coordinate transiently to the active-site zinc ion to "mask" the positive potential of the zinc ion and lower the energy barrier for dissociation of the protonated cysteine side chain from the zinc ion. In addition, a subtle conformational change by the propeptide is needed in the course of zymogen activation. These elaborate processes take place in concert in the activation process of MMPs, and the insight into these processes presented herein sheds light on a highly regulated physiological process with profound consequences for eukaryotic organisms.

Mechanism-based inhibition of zinc proteases.

The functions of zinc proteases have been implicated in a host of physiological and pathological processes in living organisms. Mechanism-based inhibitors are highly sought as biologically active molecules that afford high selectivity in targeting specific enzymes. Mechanism-based inhibitors for zinc-dependent proteases have been developed in the past several years. These inhibitors exploit the chemistry inherent to transition metals in their mechanisms of enzyme inhibition. These efforts have been reviewed in this manuscript.

X-ray absorption studies of human matrix metalloproteinase-2 (MMP-2) bound to a highly selective mechanism-based inhibitor. comparison with the latent and active forms of the enzyme.

Malignant tumors express high levels of zinc-dependent endopeptidases called matrix metalloproteinases (MMPs), which are thought to facilitate tumor metastasis and angiogenesis by hydrolyzing components of the extracellular matrix. Of these enzymes, gelatinases A (MMP-2) and B (MMP-9), have especially been implicated in malignant processes, and thus, they have been a target for drugs designed to block their activity. Therefore, understanding their molecular structure is key for a rational approach to inhibitor design. Here, we have conducted x-ray absorption spectroscopy of the full-length human MMP-2 in its latent, active, and inhibited states and report the structural changes at the zinc ion site upon enzyme activation and inhibition. We have also examined the molecular structure of MMP-2 in complex with SB-3CT, a recently reported novel mechanism-based synthetic inhibitor that was designed to be highly selective in gelatinases. It is shown that SB-3CT directly binds the catalytic zinc ion of MMP-2. Interestingly, the novel mode of binding of the inhibitor to the catalytic zinc reconstructs the conformational environment around the active site metal ion back to that of the proenzyme.

Substrate hydrolysis by matrix metalloproteinase-9.

The catalytic clefts of all matrix metalloproteinases (MMPs) have a similar architecture, raising questions about the redundancy in substrate recognition across the protein family. In the present study, an unbiased phage display strategy was applied to define the substrate recognition profile of MMP-9. Three groups of substrates were identified, each occupying a distinct set of subsites within the catalytic pocket. The most prevalent motif contains the sequence Pro-X-X-Hy-(Ser/Thr) at P(3) through P(2'). This sequence is similar to the MMP cleavage sites within the collagens and is homologous to substrates the have been selected for other MMPs. Despite this similarity, most of the substrates identified here are selective for MMP-9 over MMP-7 and MMP-13. This observation indicates that substrate selectivity is conferred by key subsite interactions at positions other than P(3) and P(1'). This study shows that MMP-9 has a unique preference for Arg at both P(2) and P(1), and a preference for Ser/Thr at P(2'). Substrates containing the consensus MMP-9 recognition motif were used to query the protein data bases. A surprisingly limited list of putative physiologic substrates was identified. The functional implications of these proteins lead to testable hypotheses regarding physiologic substrates for MMP-9.

A 1.2-A snapshot of the final step of bacterial cell wall biosynthesis.

The cell wall imparts structural strength and shape to bacteria. It is made up of polymeric glycan chains with peptide branches that are cross-linked to form the cell wall. The cross-linking reaction, catalyzed by transpeptidases, is the last step in cell wall biosynthesis. These enzymes are members of the family of penicillin-binding proteins, the targets of beta-lactam antibiotics. We report herein the structure of a penicillin-binding protein complexed with a cephalosporin designed to probe the mechanism of the cross-linking reaction catalyzed by transpeptidases. The 1.2-A resolution x-ray structure of this cephalosporin bound to the active site of the bifunctional serine type D-alanyl-D-alanine carboxypeptidase/transpeptidase (EC ) from Streptomyces sp. strain R61 reveals how the two peptide strands from the polymeric substrates are sequestered in the active site of a transpeptidase. The structure of this complex provides a snapshot of the enzyme and the bound cell wall components poised for the final and critical cross-linking step of cell wall biosynthesis.

Tethered bisubstrate derivatives as probes for mechanism and as inhibitors of aminoglycoside 3'-phosphotransferases.

Aminoglycoside 3'-phosphotransferases [APH(3')s] phosphorylate aminoglycoside antibiotics, a reaction that inactivates the antibiotics. These enzymes are the primary cause of resistance to aminoglycosides in bacteria. APH(3')-Ia operates by a random-equilibrium BiBi mechanism, whereas APH(3')-IIIa catalyzes its reaction by the Theorell-Chance mechanism, a form of ordered BiBi mechanism. Hence, both substrates have to be present in the active site prior to the transfer of phosphate by both mechanisms. Four bisubstrate analogues, compounds 1-4, were designed and synthesized as inhibitors for APH(3')s. These compounds are made of adenosine linked covalently to the 3'-hydroxyl of neamine (an aminoglycoside) via all-methylene tethers of 5-8 carbons. The K(i) values measured for these compounds indicated that affinities of APH(3')-Ia and APH(3')-IIa for compounds 2 and 3 (six- and seven-carbon tethers, respectively) were the best, and the inhibition constants for the two were comparable.

Tissue inhibitor of metalloproteinase (TIMP)-2 acts synergistically with synthetic matrix metalloproteinase (MMP) inhibitors but not with TIMP-4 to enhance the (Membrane type 1)-MMP-dependent activation of pro-MMP-2.

The membrane-type 1 matrix metalloproteinase (MT1-MMP) has been shown to be a key enzyme in tumor angiogenesis and metastasis. MT1-MMP hydrolyzes a variety of extracellular matrix components and is a physiological activator of pro-MMP-2, another MMP involved in malignancy. Pro-MMP-2 activation by MT1-MMP involves the formation of an MT1-MMP.tissue inhibitors of metalloproteinases 2 (TIMP-2). pro-MMP-2 complex on the cell surface that promotes the hydrolysis of pro-MMP-2 by a neighboring TIMP-2-free MT1-MMP. The MT1-MMP. TIMP-2 complex also serves to reduce the intermolecular autocatalytic turnover of MT1-MMP, resulting in accumulation of active MT1-MMP (57 kDa) on the cell surface. Evidence shown here in Timp2-null cells demonstrates that pro-MMP-2 activation by MT1-MMP requires TIMP-2. In contrast, a C-terminally deleted TIMP-2 (Delta-TIMP-2), unable to form ternary complex, had no effect. However, Delta-TIMP-2 and certain synthetic MMP inhibitors, which inhibit MT1-MMP autocatalysis, can act synergistically with TIMP-2 in the promotion of pro-MMP-2 activation by MT1-MMP. In contrast, TIMP-4, an efficient MT1-MMP inhibitor, had no synergistic effect. These studies suggest that under certain conditions the pericellular activity of MT1-MMP in the presence of TIMP-2 can be modulated by synthetic and natural (TIMP-4) MMP inhibitors.

Stereoselective reduction of alpha-bromopenicillanates by tributylphosphine.

[reaction: see text] Diastereoselective reduction of 6-bromo-6-substituted penicillanate esters has been achieved by treatment with tributylphosphine to give 6-substituted penicillanate esters. This reaction would appear to proceed through a phosphonium beta-lactam enolate species, followed by a diastereoselective protonation. This method has the advantage of being simple to carry out and it is mild, gives high diastereoselectivity, and should tolerate a number of functional groups in the substrates. Implications of these observations are discussed.

From genes to sequences to antibiotics: prospects for future developments from microbial genomics.

The entire genome sequences for a number of microbial organisms have become available over the past few years. This knowledge is the beginning point for understanding the fundamental principles of bacterial structure and function. The prospects for gain in knowledge from genomics are discussed in this report.

Evaluation of inhibition of the carbenicillin-hydrolyzing beta-lactamase PSE-4 by the clinically used mechanism-based inhibitors.

Characterization of the biochemical steps in the inactivation chemistry of clavulanic acid, sulbactam and tazobactam with the carbenicillin-hydrolyzing beta-lactamase PSE-4 from Pseudomonas aeruginosa is described. Although tazobactam showed the highest affinity to the enzyme, all three inactivators were excellent inhibitors for this enzyme. Transient inhibition was observed for the three inactivators before the onset of irreversible inactivation of the enzyme. Partition ratios (k(cat)/k(inact)) of 11, 41 and 131 were obtained with clavulanic acid, tazobactam and sulbactam, respectively. Furthermore, these values were found to be 14-fold, 3-fold and 80-fold lower, respectively, than the values obtained for the clinically important TEM-1 beta-lactamase. The kinetic findings were put in perspective by determining the computational models for the pre-acylation complexes and the immediate acyl-enzyme intermediates for all three inactivators. A discussion of the pertinent structural factors is presented, with PSE-4 showing subtle differences in interactions with the three inhibitors compared to the TEM-1 enzyme.

A light-inactivated antibiotic.

Sodium 7beta-[(R)-2-(N(b)()-o-nitrobenzyloxycarbonyl)hydrazino-3-pheny lpropa namido]cephalosporanate (1) is described as a new type of beta-lactam antibiotic, which undergoes light-induced destruction of its beta-lactam moiety and hence becomes biologically inactive. This type of antibiotic holds the promise of self-destruction over a number of hours of exposure to light, so that it would not allow selection of resistance in the environment.

Characterization of the monomeric and dimeric forms of latent and active matrix metalloproteinase-9. Differential rates for activation by stromelysin 1.

Matrix metalloproteinase-9 (MMP-9) is a member of the MMP family that has been associated with degradation of the extracellular matrix in normal and pathological conditions. A unique characteristic of MMP-9 is its ability to exist in a monomeric and a disulfide-bonded dimeric form. However, there exists a paucity of information on the properties of the latent (pro-MMP-9) and active MMP-9 dimer. Here we report the purification to homogeneity of the monomer and dimer forms of pro-MMP-9 and the characterization of their biochemical properties and interactions with tissue inhibitor of metalloproteinase (TIMP)-1 and TIMP-2. Gel filtration and surface plasmon resonance analyses demonstrated that the pro-MMP-9 monomeric and dimeric forms bind TIMP-1 with similar affinities. In contrast, TIMP-2 binds only to the active forms. After activation, the two enzyme forms exhibited equal catalytic competence in the turnover of a synthetic peptide substrate with comparable kinetic parameters for the onset of inhibition with TIMPs and for dissociation of the inhibited complexes. Kinetic analyses of the activation of monomeric and dimeric pro-MMP-9 by stromelysin 1 revealed K(m) values in the nanomolar range and relative low k(cat) values (1.9 x 10(-3) and 4.1 x 10(-4) s(-1), for the monomer and dimer, respectively) consistent with a faster rate (1 order of magnitude) of activation of the monomeric form by stromelysin 1. This suggests that the rate-limiting event in the activation of pro-MMP-9 may be a requisite slow unfolding of pro-MMP-9 near the site of the hydrolytic cleavage by stromelysin 1.