A Unified Numbering Scheme for Class C β-Lactamases

A standard numbering scheme has been proposed for class C β-lactamases. This will significantly enhance comparison of biochemical and biophysical studies performed on different members of this class of enzymes and facilitate communication in the field.

ABSTRACT

A standard numbering scheme has been proposed for class C β-lactamases. This will significantly enhance comparison of biochemical and biophysical studies performed on different members of this class of enzymes and facilitate communication in the field.

TEXT

β-Lactamases hydrolyze β-lactam antibiotics, such as penicillins, cephalosporins, and carbapenems, rendering them inactive, and thus, they represent one of the most important mechanisms of bacterial resistance toward these agents (1). These enzymes have been classified according to substrate and inhibitor specificity (1–3) and according to their primary protein sequence (4). Four molecular classes are generally recognized, A, B, C, and D (5): enzymes in classes A, C, and D have a serine residue in the catalytic center that is activated by neighboring residues, and perhaps a substrate moiety, to carry out nucleophilic attack on the carbonyl moiety of the β-lactam, forming an acyl ester at rates that approach limitation by the rate of diffusion (6, 7). The acyl ester is subsequently hydrolyzed by a water molecule, also activated by residues in the catalytic center, at a lower rate that may be governed by rearrangement of the enzyme complex (8–10). The three classes of serine enzymes differ in their molecular structures and in mechanisms of carrying out the hydrolysis of the acyl ester (11).

The class C β-lactamases comprise the second most abundant group of enzymes and are found solely in Gram-negative bacteria, especially members of the Enterobacteriaceae, Pseudomonas aeruginosa, and Acinetobacter species (1, 12). Although they were originally identified as chromosomally encoded, more recently, genes encoding class C enzymes have been found mobilized on plasmids in the Enterobacteriaceae (13). The class C enzymes fall into group 1 of the Bush-Jacoby-Medeiros functional classification and are recognized as cephalosporinases because of the high turnover numbers exhibited by many of the members of this class for first- and second-generation cephalosporins and cephamycins. The catalytic efficiency (k_cat/K_m) is, however, rather similar for penicillins, for which many of the enzymes have high apparent affinities (low K_m) but correspondingly low turnover numbers. The enzymes in this class generally exhibit low turnover numbers for third-generation cephalosporins and very low turnover numbers for monobactams and carbapenems, although they react readily with these agents to form rather stable acyl-enzyme intermediates (14). Despite the low turnover, the enzymes can still afford protection against these agents, partly because the enzymes can be expressed to very high levels in the periplasmic space of Gram-negative bacteria, where they may act as a sink, trapping the antibiotic before it can reach its lethal target, and partly because the resistant bacteria have acquired additional mechanisms of resistance that restrict the net influx of the antibiotic (15). These additional mechanisms can involve mutation of residues in the outer membrane porins (16), mutations in regulatory systems that result in low expression of these proteins, and loss of the porin genes, as well as mutations in the tripartite RND family efflux systems, or their regulatory systems, that result in increased activity of these pumps (17, 18).

The mechanism by which the class C enzymes hydrolyze β-lactam antibiotics is still incompletely understood. From the crystal structure of the complex formed between aztreonam and the Citrobacter freundii chromosomal enzyme, it has been concluded that a tyrosine residue in the catalytic center acts a general base during hydrolysis, activating both the serine residue for attack on the β-lactam nucleus and a water molecule for attack on the ester bond formed between the serine and the lactam moiety (19). Subsequently, further complexes of class C enzymes from different species with a variety of substrates and inhibitors have become available, shedding further light on the details of the acylation mechanism (14). Additional studies using site-directed mutagenesis to explore the contributions of selected residues in and around the catalytic center have added further understanding of the mechanism (20–25). Kinetic studies, including time-resolved Fourier-transform infrared spectroscopy studies, have shown that there are significant conformational changes during the catalytic cycle, changes that are not obvious in the time frame of the crystallographic studies (9, 10).

Bringing these disparate studies together requires a consistent frame of reference: the studies on isolated proteins will have residue numbers starting from the first amino acid in the primary sequence of the isolated protein, whereas genetic studies may refer to numbers starting from the first codon in the translated gene sequence (26, 27) and these may differ from numbering derived from multiple sequence alignment of naturally occurring variants within a species (28). There is therefore an urgent need to address the inconsistencies between numbering of amino acid residues in different studies on the same enzyme. Despite the class C enzyme from Escherichia coli being the first β-lactamase to be recorded (29), the nomenclature of this group has lagged behind advances in nomenclature in other families (30). Workers studying the class A β-lactamases have benefitted for nearly 3 decades from a unified standard numbering scheme that fixes the positions of certain important residues and allows for insertion or deletion of residues at other positions in the sequence (5), and an analogous scheme for numbering residues of class B enzymes was proposed nearly 20 years ago (31). In this issue, Andrew Mack and colleagues (32) present a similar standard numbering system for the class C β-lactamase family. The proposal is based on multiple sequence alignment of 32 sequences of class C enzymes, including both chromosomal and plasmid-encoded proteins, and derivation of a consensus secondary structure map from the 10 class C β-lactamases for which an X-ray crystal structure in available in the Protein Data Bank. The proposed amino acid numbering system is based on the sequence of Enterobacter cloacae P99 and keeps the widely followed conventional numbering of the important residues in the catalytic center: serine 64, lysine 67, tyrosine 150, and lysine 315. In the proposed scheme, any insertions relative to the reference sequence are denoted by appending a lowercase letter (e.g., “a”) to the number of the residue immediately preceding the insertion, while any deletions are skipped (e.g., ACC-1 has the sequence G115 followed by L117 due to an apparent deletion at position 116 in comparison to the reference sequence). For mature enzymes that are longer than the reference sequence, the first residue of an N-terminal extension is denoted “0” and any further extension is denoted by adding a lowercase letter (e.g., “a”), while any additional residues at the C terminus are assigned increasing numbers. Residues in signal peptides are identified by negative numbers starting with −1 at the (predicted) cleavage site and decreasing toward the N terminus of the peptide. Examples illustrating the application of these very logical rules are given in the article. The authors suggest that the standard numbering scheme should be used in biochemical and biophysical publications but that the usual convention of starting at residue 1 for the signal sequence should be followed in genetic and epidemiological studies. The adoption of this standard numbering will considerably reduce complications in communication in the field and is to be much encouraged.

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