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Biomedical subjects

S Mobashery

Publications and source records attributed to S Mobashery.

At least 37 records · Page 2Linked to original sources
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↗
PubMed1998

The use of neamine as a molecular template: identification of active site residues in the bacterial antibiotic resistance enzyme aminoglycoside 3'-phosphotransferase type IIa by mass spectroscopy.

Four novel aminoglycoside-based affinity inactivators were shown to covalently modify the active site of aminoglycoside 3'-phosphotransferase type IIa (APH(3')-IIa), an important resistance factor in bacteria for aminoglycoside antibiotics. Standard peptide mapping techniques failed with this enzyme. A novel mass spectroscopic analysis which combines protease digestion on the instrument probe, followed by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) is described which permitted rapid identification of the sites of protein modification. By this new technique, Glu-3 and Asp-23 were identified as active-site residues, the side chains of which potentially may serve as counter ions for the ammonium functionalities at positions 6', and 1 and 3 of the antibiotic substrates, respectively. These findings contradict previous assertions that the C-terminal third of the enzyme should form the active site, by placing the active site clearly in the N-terminal portion of the enzyme.

Aspartic Acid↗
PubMed1998

Templates for design of inhibitors for serine proteases: application of the program DOCK to the discovery of novel inhibitors for thrombin.

The program DOCK was used to search for novel inhibitors for alpha-thrombin. Four among the top twelve best scoring compounds from the Cambridge Structural Data Base inhibited this enzyme, and three of them inhibited alpha-thrombin in a competitive mode. These molecules are expected to serve as general templates for structural elaboration in targeting diverse serine proteases for selective inhibition.

Binding Sites↗
PubMed1998

Selection and characterization of beta-lactam-beta-lactamase inactivator-resistant mutants following PCR mutagenesis of the TEM-1 beta-lactamase gene.

Mechanism-based inactivators of beta-lactamases are used to overcome the resistance of clinical pathogens to beta-lactam antibiotics. This strategy can itself be overcome by mutations of the beta-lactamase that compromise the effectiveness of their inactivation. We used PCR mutagenesis of the TEM-1 beta-lactamase gene and sequenced the genes of 20 mutants that grew in the presence of ampicillin-clavulanate. Eleven different mutant genes from these strains contained from 1 to 10 mutations. Each had a replacement of one of the four residues, Met69, Ser130, Arg244, and Asn276, whose substitutions by themselves had been shown to result in inhibitor resistance. None of the mutant enzymes with multiple amino acid substitutions generated in this study conferred higher levels of resistance to ampicillin alone or ampicillin with beta-lactamase inactivators (clavulanate, sulbactam, or tazobactam) than the levels of resistance conferred by the corresponding single-mutant enzymes. Of the four enzymes with just a single mutation (Ser130Gly, Arg244Cys, Arg244Ser, or Asn276Asp), the Asn276Asp beta-lactamase conferred a wild-type level of ampicillin resistance and the highest levels of resistance to ampicillin in the presence of inhibitors. Site-directed random mutagenesis of the Ser130 codon yielded no other mutant with replacement of Ser130 besides Ser130Gly that produced ampicillin-clavulanate resistance. Thus, despite PCR mutagenesis we found no new mutant TEM beta-lactamase that conferred a level of resistance to ampicillin plus inactivators greater than that produced by the single-mutation enzymes that have already been reported in clinical isolates. Although this is reassuring, one must caution that other combinations of multiple mutations might still produce unexpected resistance.

Ampicillin↗
PubMed1998

Matrix metalloproteinases: structures, evolution, and diversification.

A comprehensive sequence alignment of 64 members of the family of matrix metalloproteinases (MMPs) for the entire sequences, and subsequently the catalytic and the hemopexin-like domains, have been performed. The 64 MMPs were selected from plants, invertebrates, and vertebrates. The analyses disclosed that as many as 23 distinct subfamilies of these proteins are known to exist. Information from the sequence alignments was correlated with structures, both crystallographic as well as computational, of the catalytic domains for the 23 representative members of the MMP family. A survey of the metal binding sites and two loops containing variable sequences of amino acids, which are important for substrate interactions, are discussed. The collective data support the proposal that the assembly of the domains into multidomain enzymes was likely to be an early evolutionary event. This was followed by diversification, perhaps in parallel among the MMPs, in a subsequent evolutionary time scale. Analysis indicates that a retrograde structure simplification may have accounted for the evolution of MMPs with simple domain constituents, such as matrilysin, from the larger and more elaborate enzymes.

Amino Acid Sequence↗
PubMed1997

Kinetic analysis of the binding of human matrix metalloproteinase-2 and -9 to tissue inhibitor of metalloproteinase (TIMP)-1 and TIMP-2.

The dissociation constants (Kd) of tissue inhibitor of metalloproteinase (TIMP)-1 and TIMP-2 for the active and latent forms of matrix metalloproteinase (MMP)-2 and MMP-9 were evaluated using surface plasmon resonance (SPR) and enzyme inhibition studies. SPR analysis shows biphasic kinetics with high (nM) and low (microM) affinity binding sites of TIMP-2 and TIMP-1 for MMP-2 (72- and 62-kDa species) and MMP-9 (92- and 82-kDa species), respectively. In contrast, binding data of TIMP-2 to an MMP-2 45-kDa active form lacking the C-terminal domain and to an MMP-2 C-terminal domain (CTD) fragment displays monophasic kinetics with Kd values of 315 and 60 nM, respectively. This suggests that the CTD contains the high affinity binding site, whereas the catalytic domain contains the low affinity site. Also, binding of TIMP-2 to pro-MMP-2 is stronger at both the high and low affinity sites than the corresponding binding of TIMP-2 to the MMP-2 62-kDa form demonstrating the importance of the N-terminal prodomain. In addition, the Kd value of TIMP-1 for the MMP-2 62-kDa species is 28. 6 nM at the high affinity site, yet neither the MMP-2 45-kDa species nor the CTD interacts with TIMP-1. Enzyme inhibition studies demonstrate that TIMPs are slow binding inhibitors with monophasic inhibition kinetics. This suggests that a single binding event results in enzyme inhibition. The kinetic parameters for the onset of inhibition are fast (kon approximately 10(5) M-1 s-1) with slow off rates (koff approximately 10(-3) s-1). The inhibition constants (Ki) are in the 10(-7)-10(-9) M range and correlate with the values determined by SPR.

Biosensing Techniques↗