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S Mobashery

Publications and source records attributed to S Mobashery.

At least 19 recordsLinked to original sources
PubMed2001

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.

Acylation↗
PubMed2000

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.

Animals↗
PubMed2000

Insights into class D beta-lactamases are revealed by the crystal structure of the OXA10 enzyme from Pseudomonas aeruginosa.

BACKGROUND: beta-lactam antibiotic therapies are commonly challenged by the hydrolytic activities of beta-lactamases in bacteria. These enzymes have been grouped into four classes: A, B, C, and D. Class B beta-lactamases are zinc dependent, and enzymes of classes A, C, and D are transiently acylated on a serine residue in the course of the turnover chemistry. While class A and C beta-lactamases have been extensively characterized by biochemical and structural methods, class D enzymes remain the least studied despite their increasing importance in the clinic. RESULTS: The crystal structure of the OXA10 class D beta-lactamase has been solved to 1.66 A resolution from a gold derivative and MAD phasing. This structure reveals that beta-lactamases from classes D and A, despite very poor sequence similarity, share a similar overall fold. An additional beta strand in OXA10 mediates the association into dimers characterized by analytical ultracentrifugation. Major differences are found when comparing the molecular details of the active site of this class D enzyme to the corresponding regions in class A and C beta-lactamases. In the native structure of the OXA10 enzyme solved to 1.8 A, Lys-70 is carbamylated. CONCLUSIONS: Several features were revealed by this study: the dimeric structure of the OXA10 beta-lactamase, an extension of the substrate binding site which suggests that class D enzymes may bind other substrates beside beta-lactams, and carbamylation of the active site Lys-70 residue. The CO2-dependent activity of the OXA10 enzyme and the kinetic properties of the natural OXA17 mutant protein suggest possible relationships between carbamylation, inhibition of the enzyme by anions, and biphasic behavior of the enzyme.

Amino Acid Sequence↗
PubMed2000

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.

Carbohydrate Sequence↗
PubMed2000

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.

Esters↗
PubMed2000

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.

Acylation↗
PubMed2000

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.

Amino Acid Sequence↗
PubMed2000

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.

Bacteria↗
PubMed1999

Inhibition of the broad spectrum nonmetallocarbapenamase of class A (NMC-A) beta-lactamase from Enterobacter cloacae by monocyclic beta-lactams.

beta-Lactamases hydrolyze beta-lactam antibiotics, a reaction that destroys their antibacterial activity. These enzymes, of which four classes are known, are the primary cause of resistance to beta-lactam antibiotics. The class A beta-lactamases form the largest group. A novel class A beta-lactamase, named the nonmetallocarbapenamase of class A (NMC-A) beta-lactamase, has been discovered recently that has a broad substrate profile that included carbapenem antibiotics. This is a serious development, since carbapenems have been relatively immune to the action of these resistance enzymes. Inhibitors for this enzyme are sought. We describe herein that a type of monobactam molecule of our design inactivates the NMC-A beta-lactamase rapidly, efficiently, and irreversibly. The mechanism of inactivation was investigated by solving the x-ray structure of the inhibited NMC-A enzyme to 1.95 A resolution. The structure shed light on the nature of the fragmentation of the inhibitor on enzyme acylation and indicated that there are two acyl-enzyme species that account for enzyme inhibition. Each of these inhibited enzyme species is trapped in a distinct local energy minimum that does not predispose the inhibitor species for deacylation, accounting for the irreversible mode of enzyme inhibition. Molecular dynamics simulations provided evidence in favor of a dynamic motion for the acyl-enzyme species, which samples a considerable conformational space prior to the entrapment of the two stable acyl-enzyme species in the local energy minima. A discussion of the likelihood of such dynamic motion for turnover of substrates during the normal catalytic processes of the enzyme is presented.

Anti-Bacterial Agents↗
PubMed1999

Effects on substrate profile by mutational substitutions at positions 164 and 179 of the class A TEM(pUC19) beta-lactamase from Escherichia coli.

We investigated the effects of mutations at positions 164 and 179 of the TEM(pUC19) beta-lactamase on turnover of substrates. The direct consequence of some mutations at these sites is that clinically important expanded-spectrum beta-lactams, such as third-generation cephalosporins, which are normally exceedingly poor substrates for class A beta-lactamases, bind the active site of these mutant enzymes more favorably. We employed site-saturation mutagenesis at both positions 164 and 179 to identify mutant variants of the parental enzyme that conferred resistance to expanded-spectrum beta-lactams by their enhanced ability to turn over these antibiotic substrates. Four of these mutant variants, Arg(164) --> Asn, Arg(164) --> Ser, Asp(179) --> Asn, and Asp(179) --> Gly, were purified and the details of their catalytic properties were examined in a series of biochemical and kinetic experiments. The effects on the kinetic parameters were such that either activity with the expanded-spectrum beta-lactams remained unchanged or, in some cases, the activity was enhanced. The affinity of the enzyme for these poorer substrates (as defined by the dissociation constant, K(s)) invariably increased. Computation of the microscopic rate constants (k(2) and k(3)) for turnover of these poorer substrates indicated either that the rate-limiting step in turnover was the deacylation step (governed by k(3)) or that neither the acylation nor deacylation became the sole rate-limiting step. In a few instances, the rate constants for both the acylation (k(2)) and deacylation (k(3)) of the extended-spectrum beta-lactamase were enhanced. These results were investigated further by molecular modeling experiments, using the crystal structure of the TEM(pUC19) beta-lactamase. Our results indicated that severe steric interactions between the large 7beta functionalities of the expanded-spectrum beta-lactams and the Omega-loop secondary structural element near the active site were at the root of the low affinity by the enzyme for these substrates. These conclusions were consistent with the proposal that the aforementioned mutations would enlarge the active site, and hence improve affinity.

Amino Acid Substitution↗
PubMed1999

X-ray structure of the Asn276Asp variant of the Escherichia coli TEM-1 beta-lactamase: direct observation of electrostatic modulation in resistance to inactivation by clavulanic acid.

The clinical use of beta-lactam antibiotics combined with beta-lactamase inactivators, such as clavulanate, has resulted in selection of beta-lactamases that are insensitive to inactivation by these molecules. Therefore, therapeutic combinations of an enzyme inactivator and a penicillin are harmless for bacteria harboring such an enzyme. The TEM beta-lactamase variants are the most frequently encountered enzymes of this type, and presently, 20 variants are designated as inhibitor-resistant TEM ("IRT") enzymes. Three mutations appear to account for the phenotype of the majority of IRT enzymes, one of them being the Asn276Asp substitution. In this study, we have characterized the kinetic properties of the inhibition process of the wild-type TEM-1 beta-lactamase and of its Asn276Asp variant with the three clinically used inactivators, clavulanic acid (clavulanate), sulbactam, and tazobactam, and we report the X-ray structure for the mutant variant at 2.3 A resolution. The changes in kinetic parameters for the interactions of the inhibitors with the wild-type and the mutant enzymes were more pronounced for clavulanate, and relatively inconsequential for sulbactam and tazobactam. The structure of the Asn276Asp mutant enzyme revealed a significant movement of Asp276 and the formation of a salt bridge of its side chain with the guanidinium group of Arg244, the counterion of the inhibitor carboxylate. A water molecule critical for the inactivation chemistry by clavulanate, which is observed in the wild-type enzyme structure, is not present in the crystal structure of the mutant variant. Such structural changes favor the turnover process over the inactivation chemistry for clavulanate, with profound phenotypic consequences. The report herein represents the best studied example of inhibitor-resistant beta-lactamases.

Asparagine↗
PubMed1999

Class C beta-lactamases operate at the diffusion limit for turnover of their preferred cephalosporin substrates.

It has been suggested that class C beta-lactamases have evolved to carry out a metabolic reaction other than hydrolysis of beta-lactam antibiotics. It is demonstrated in the present study that the class C beta-lactamase from Enterobacter cloacae P99 has reached the diffusion limit in its ability to hydrolyze its preferred cephalosporin substrates. The increase in the solution viscosity by addition of a microviscogen (sucrose) caused the decline in the parameter kcat/Km for hydrolysis of cephaloridine and cephalosporin C (approximately 2.5-fold at a relative viscosity of 2.9). A similar increase in viscosity has no effect on the turnover rate of the poorer substrates cefepime and penicillin G. Addition of a macroviscogen (polyethylene glycol) to the reaction mixture did not change the rate of turnover for any of the substrates tested because in this case the viscogen would not interfere with the motion of small molecules, as was expected. Therefore, it would appear that the driving force behind the evolution of this class C beta-lactamase and, in principle, other enzymes of this class is indeed the functional reaction of this enzyme as a drug resistance factor.

Cephalosporins↗
PubMed1999

Mechanistic and clinical aspects of beta-lactam antibiotics and beta-lactamases.

Bacterial infections have been a major cause of concern in the recent years due to the emergence of drug resistance strains and inability of the current therapeutic regimens to treat these infections in certain cases. Beta-Lactam antibiotics have been drugs of choice since the introduction of penicillin. These drugs inhibit bacterial cell-wall-synthesizing enzymes, the so-called penicillin-binding proteins (PBPs) selectively, thus providing an effective strategy for treatment of the bacterial infections. Significantly, bacteria have developed resistance mechanisms to neutralize the antibiotic action of beta-lactam drugs. Beta-Lactamases are enzymes that hydrolyze the beta-lactam moiety of these drugs, rendering them inactive. This is the primary mechanism of resistance to this class of antibiotics. There are 255 known beta-lactamases to date and the continued use of beta-lactams may select for newer variants yet. A discussion of the roles of these enzymes in the manifestation of the drug-resistant phenotype and their implications for pathogenecity of clinical strains of bacteria is presented.

Anti-Bacterial Agents↗
PubMed1999

Structural and mechanistic aspects of evolution of beta-lactamases and penicillin-binding proteins.

Penicillin-binding proteins (PBPs) and b-lactamases are related enzymes, the former are the targets for b-lactam antibiotics and the latter are resistance enzyme to these antibiotics. The two families of enzymes share structural topologies and certain mechanistic features. However, these classes of enzymes have diversified substantially and have broadened the reaction repertoire for their catalytic properties. This report addresses the issues of the evolution of function of these proteins.

Acylation↗
PubMed1998

The use of neamine as a molecular template: inactivation of bacterial antibiotic resistance enzyme aminoglycoside 3'-phosphotransferase type IIa.

Aminoglycoside 3'-phosphotransferase type IIa [APH(3')-IIa] is a member of the family of bacterial aminoglycoside-modifying enzymes. Bacteria that harbor these enzymes are resistant to aminoglycoside antibiotics. Four aminoglycoside-based affinity inactivators were synthesized and were shown to be both substrates and inactivators for APH(3')-IIa. These affinity inactivators are N-bromoacetylated derivatives of neamine, an aminoglycoside antibiotic, where the bromoacetyl moiety in each was introduced regiospecifically at a different amine of the parent compound.

Bacteria↗