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Volume 5, Number 11, 1999: Contents

b-Lactamases of Increasing Clinical Importance. Pp. 839-845.
Karen Bush
[Abstract]

SHV-Type b-Lactamases. Pp. 847-864.
Leonidas S. Tzouvelekis and Robert A. Bonomo
[Abstract]

OXA-Type b-Lactamases. Pp. 865-879.
Thierry Naas and Patrice Nordmann
[Abstract]

Regulation of Inducible AmpC Beta-Lactamase Expression Among Enterobacteriaceae. Pp. 881-894.
N.D. Hanson and C.C. Sanders
[Abstract]

The Reactivity of b-Lactams, the Mechanism of Catalysis and the Inhibition of b-Lactamases. Pp. 895-913.
Michael I. Page
[Abstract]

Class B b-Lactamases: the Importance of Being Metallic. Pp. 915-927.
Julia A. Cricco and Alejandro J. Vila
[Abstract]

Structural and Mechanistic Aspects of Evolution of b-Lactamases and Penicillin-Binding Proteins. Pp. 929-937.
Irina Massova and Shahriar Mobashery
[Abstract]

Recent Advances in the Chemistry of b-Lactam Compounds as Selected Active-site Serine b-Lactamase Inhibitors. Pp. 939-953.
Oreste A. Mascaretti, Gerardo O. Danelon, Eduardo L. Setti, María Laborde and Ernesto G. Mata
[Abstract]

Solid-phase and Combinatorial Synthesis in b-Lactam Chemistry. Pp. 955-964.
Ernesto G. Mata
[Abstract]


Abstracts

[Back to top] b-Lactamases of Increasing Clinical Importance. Karen Bush.
Resistance to b-Lactam-containing antimicrobial agents continues to increase, frequently due to the presence of b-Lactamases in Gram-negative bacteria. Over the past twenty-five years broad-spectrum enzymes such as TEM- and SHV-variants and the metallo-b-Lactamases have become more prolific. As a result of the ability of plasmids to continue to acquire additional resistance determinants, many of the b-Lactamase-producing Gram-negative pathogens have become multi-drug resistant. In combination with decreased permeability, the organisms can become virtually untreatable with current therapies. The major groups of b-Lactamases that pose the most serious therapeutic problems include the extended-spectrum b-Lactamases, the plasmid-mediated cephalosporinases, the inhibitor-resistant TEM- or SHV-derived b-Lactamases and the carbapenem-hydrolyzing b-Lactamases. Those enzymes that can be transferred on mobile elements are the most serious of the newer b-Lactamases, and include enzymes in each of the four groups outlined above.

[Back to top] SHV-Type b-Lactamases. Leonidas S. Tzouvelekis and Robert A. Bonomo.
The group of plasmid-mediated SHV b-Lactamases includes SHV-1 and at least twenty-three variants, most of which possess extended-spectrum (ES) activity against the newer broad-spectrum cephalosporins. Their likely ancestor is a chromosomal penicillinase of Klebsiella pneumoniae. SHV enzymes belong to the molecular class A of serine b-Lactamases and share extensive functional and structural similarity with TEM b-Lactamases. The three-dimensional structure of the SHV-1 b-Lactamase possesses an active site wider than that of TEM-1 b-Lactamase by 0.7 to 1.2 Å. This results in subtle, yet important, differences in the positioning of critical active-site residues. SHV-1 b-Lactamase behaves as a typical penicillinase hydrolyzing penicillins and early generation cephalosporins. SHV-1 b-Lactamase has spread, via plasmids, to virtually all enterobacterial species but is encountered mostly in K. pneumoniae. ES SHV b-Lactamases are found with increasing frequency in K. pneumoniae and other enterobacterial isolates and are now considered the most prevalent ES b-Lactamases. These ES SHV b-Lactamases confer a wide spectrum of resistance to b-Lactams, including the new generation cephalosporins and monobactams, and are usually encoded by self-transmissible multi-resistant plasmids that are highly mobile. Extension of the hydrolytic spectrum of ES SHV enzymes to include oximino-b-Lactams is seen as a result of substitutions of critical amino acid residues that alter the properties of the active site. These mutational changes, however, result in diminished hydrolytic activity against penicillins and an increased susceptibility to mechanism-based inhibitors. Understanding the substrate evolution, properties and modes of spread of these clinically important b-Lactamases can help in formulating effective antibiotic policies and developing new antimicrobial agents.

[Back to top] OXA-Type b-Lactamases. Thierry Naas and Patrice Nordmann.
The OXA-type (oxacillin-hydrolysing) enzymes are widespread and have been mostly described in Enterobacteriaceae and in P. aeruginosa. They usually confer resistance to amino- and ureidopenicillin and possess high-level hydrolytic activity against cloxacillin, oxacillin, and methicillin. Their activities are weakly inhibited by clavulanic acid but sodium chloride (NaCl) possesses a strong inhibition activity. Oxacillin-hydrolysing b-Lactamases belong to Ambler class D and thus possess an active serine site as classes A and C b-Lactamases. Overall amino-acid identities between class D and class A or class C b-Lactamases is about 16%. Until now, 24 Ambler class D enzymes, named OXA-1 to OXA-22, AmpS and LCR-1, have been characterised, either by sequence and/or by biochemical analyses, but for none of them a three dimensional structure is yet available. While some oxacillinases present a significant degree of amino-acid identity (for example, OXA-1 and OXA-4; OXA-10 (PSE-2) derivatives; OXA-2 and OXA-3), most of them are only weakly related (20% to 30% amino-acid identity). Oxacillinases usually display a restricted-spectrum phenotype. However extension of their spectrum towards oxyimino cephalosporins and/or imipenem has recently been observed mostly as a consequence of point mutations in OXA-2 or OXA-10 derivatives. Their frequent plasmid- and/or integron-location provide them a mean for a wide diffusion.

[Back to top] Regulation of Inducible AmpC Beta-Lactamase Expression Among Enterobacteriaceae. N.D. Hanson and C.C. Sanders.
AmpC b-lactamases are active-site serine enzymes that are primarily cephalosporinases. In many gram negative organisms, including Enterobacter spp.,Citrobacter freundii, Serratia marcescens, Morganella morganii and Pseudomonas aeruginosa, the expression of chromosomal ampC genes is low but inducible in response to b-lactams and other stimuli. The current working model for AmpC induction requires exposure of bacterial cells to b-lactam drugs or other stimuli and is linked to the cell wall recycling pathway. Induction of ampC appears to involve several gene products associated with this pathway. These gene products include AmpR, AmpD, and AmpG. In addition, anhydro forms of cell wall precursor muropeptides are believed to act as cofactors for AmpC induction. These cofactors bind to the DNA binding protein, AmpR, and define the role of AmpR as activator. Recent debate has ensued in the literature as to the identification of the precursor muropeptide involved in the activation process. Two candidate muropeptides include 1,6-anhydro-N-acetylmuramic acid L-Ala-D-Glu-meso-diaminopimelic acid (anhydro-MurNAc-tripeptide) and anhydro-MurNAc-L-Ala-D-Glu-meso-diaminopimelic acid- D-Ala-D-Ala (pentapeptide). The intent of this review is to address the general mechanism involved in AmpC induction. In doing so, the genes and gene products required for the process of AmpC induction are described. In addition, we review the data addressing cell wall recycling as it relates to AmpC induction.

[Back to top] The Reactivity of b-Lactams, the Mechanism of Catalysis and the Inhibition of b-Lactamases. Michael I. Page.
Four membered b-Lactam rings do not show unusual reactivity compared with their acyclic amide analogues and there is no evidence of concerted mechanisms for nucleophilic substitution reactions at the carbonyl centre. The identity of the general base/acid catalyst in the serine b-Lactamases, which catalyse the hydrolysis of b-Lactams, is unknown. There are no ideal transition state analogue inhibitors for these enzymes which involve several intermediates and transition states. The class C serine b-Lactamase enhances the rate of phosphonylation of its active site serine residue by a similar magnitude to the enzyme rate enhancement factor for the hydrolysis of b-Lactams. Comparisons are made between the stereochemical consequences of tetrahedral and trigonal bipyramidal intermediates for hydrolysis and phosphonylation respectively. Class B zinc b-Lactamases are inhibited by thiol dipeptides with a D configuration at the cysteine centre analogous to the L configuration at C6 in penicillins. The mechanism of hydrolysis catalysed by the metallo-b-Lactamases probably involves a di-anionic tetrahedral intermediate stabilised by zinc(II).

[Back to top] Class B b-Lactamases: the Importance of Being Metallic. Julia A. Cricco and Alejandro J. Vila.
The structural and functional features of class B b-Lactamases, which are metal-dependent, are reviewed in this article. Enzymes from different bacterial strains exhibit a common fold and sequence similarity in their active sites. However, the protein scaffold fine tunes the metal binding affinity and substrate selectivity. In this way, some metallo-b-Lactamases seem to be functional with only one Zn(II) equivalent per enzyme, whereas others require a binuclear active site. The sequence similarity leads to a subdivision of these enzymes into three subclasses. The substrate specificities are rather broad, except for enzymes belonging to subclass B2. Some inhibitors have been designed and tested, but none of them is able to exhibit a broad spectrum against these enzymes.

[Back to top] Structural and Mechanistic Aspects of Evolution of b-Lactamases and Penicillin-Binding Proteins. Irina Massova and Shahriar Mobashery.
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.

[Back to top] Recent Advances in the Chemistry of b-Lactam Compounds as Selected Active-site Serine b-Lactamase Inhibitors. Oreste A. Mascaretti, Gerardo O. Danelon, Eduardo L. Setti, María Laborde and Ernesto G. Mata.
The b-Lactamases catalyze the hydrolysis of the b-Lactam bond of a variety of b-Lactam antibiotics destroying their antibacterial activity. During the last decades, there has been an inexorable spread of b-Lactamase genes into species that previously were not known to possess them.

One approach to combat the action of the b-Lactamase is to inhibit the enzyme. However, inhibition of b-Lactamase alone is not sufficient. The ability to penetrate the external membrane of Gram-negative bacteria, chemical stability, pharmacokinetics and other factors are also important in determining whether an inhibitor is suitable or not for therapeutic use.

This review takes recent examples of synthetic b-Lactam compounds developed as active-site serine b-Lactamase inhibitors, emphasizing information on their structures and their activity against Ambler classes A, C and D b-Lactamases. In addition, examples based on rational design by computerized molecular modeling of crystal structure of the native enzyme and mechanism of the enzyme action are highlighted.

[Back to top] Solid-phase and Combinatorial Synthesis in b-Lactam Chemistry. Ernesto G. Mata.
Combinatorial chemistry has became a core technology for the rapid development of novel lead compounds in the pharmaceutical industry and for the optimization of therapeutic efficacy. The effort to prepare libraries of compounds by combinatorial chemistry has led to an unprecedented growth in solid phase organic synthesis (SPOS), particularly for the preparation of non-oligomeric small molecules. In this context, the clinically valuable b-Lactam compounds are very attractive targets for research using these new techniques. In recent years, b-Lactam compounds have been recognized not only as unique antibacterial agents but also as potent enzyme inhibitors, drug delivery agents, and versatile synthetic intermediates. This review gives a comprehensive up-date for the application of solid-phase and combinatorial synthesis to b-Lactam compounds.