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Antimicrobial Agents and Chemotherapy, July 2004, p . 2347-2349, Vol . 48, No . 7

Update of the Standard Numbering Scheme for Class B ß-Lactamases

Gianpiero Garau,1 Isabel García-Sáez,1 Carine Bebrone,2 Christine Anne,2 Paola Mercuri,2 Moreno Galleni,2 Jean-Marie Frère,2 and Otto Dideberg1*

Laboratoire de Cristallographie Macromoléculaire, Institut de Biologie Structurale "Jean-Pierre Ebel," CEA-CNRS-UJF, F-38027 Grenoble, France,1 Centre d'Ingénierie des Protéines, Laboratoire d'Enzymologie, Institut de Chimie, Université de Liège, B6 Sart-Tilman, B-4000 Liège, Belgium2


   TEXT

 
ß-Lactamases represent the major cause of bacterial resistance against ß-lactam antibiotics, and they have been divided into four classes (A to D) on the basis of their amino acid sequences (21) . The class B enzymes have no sequence or structural similarity to the active-site serine enzymes of classes A, C, and D (6); require a bivalent metal ion (Zn2+) for activity; and constitute group 3 in the Bush-Jacoby-Medeiros functional classification (2) . The identification of Zn-ß-lactamase-producing pathogenic strains of Aeromonas, Bacteroides, Flavobacterium, Legionella, Serratia, and Stenotrophomonas has greatly increased interest in this class of enzymes (2) . The fact that they hydrolyze almost all ß-lactam antibiotics, including carbapenems, underlines their clinical relevance . In consequence, the potential spreading of these enzymes among pathogenic bacteria is a frightening possibility, which emphasizes the importance of understanding their properties .

On the basis of the sequences, three subclasses of class B ß-lactamases (B1 to B3) were identified, and a standard numbering scheme (BBL numbering) was proposed (13) by analogy to the ABL numbering scheme which has been widely used for class A ß-lactamases . Due to the general low degree of identity between subclass sequences (<20%), classical alignment programs produce unreliable results . The proposed alignment (13) was facilitated by the availability of X-ray structures for B1 and B3 enzymes . Crystallographic structures have been described for several B1 enzymes: Bacillus cereus BcII (4, 11), Bacteroides fragilis CcrA (5, 8), Pseudomonas aeruginosa IMP-1 (7) and VIM-2 (unpublished data), and Chryseobacterium meningosepticum BlaB (14) . Structural data are also available for two B3 enzymes: Stenotroptromonas maltophilia L1 (28) and Legionella gormanii FEZ-1 (15) . Recently, we solved the first X-ray structure of a subclass B2 enzyme (CphA) produced by various species of Aeromonas (G . Garau, C . Bebrone, C . Anne, M . Galleni, J.-M . Frère, and O . Dideberg, unpublished data) . Using all available three-dimensional structures, it is now possible to propose a bonafide structural alignment of the class B ß-lactamases, and accordingly, to update the first proposed BBL scheme (Fig . 1) .


FIG . 1 . Structural alignment of eight class B ß-lactamases with known X-ray structures . The sequences are referred to by their familiar names . BCII, B . cereus 569H (16); IMP-1, P . aeruginosa 101/477 (17); CcrA, B . fragilis TAL3636 (25); VIM-2, P . aeruginosa species (24); BlaB, C . meningosepticum NCTC10585 (26); CphA, Aeromonas hydrophila AE036 (20); L1, S . maltophilia IID1275 (29); FEZ-1, L . gormanii ATCC33297T (1) . The BBL numbering is defined . Conserved secondary-structure elements for the three subclasses are indicated above the sequences: S, ß-strand; H, helix; L, loop . Amino acid insertions in newly sequenced enzymes are represented by lowercase letters . The zinc ligands in at least one subclass are shaded and labeled as follows: Z, residues conserved in the three subclasses; •, residues conserved in subclass B1 and some enzymes of subclass B3; +, residue conserved in subclass B3; §, residues conserved in subclasses B1 and B2 . L2 and L4 represent largely variable regions . The 14 sequence fragments of structurally conserved positions, which cover the entirety of all sequences, are shown in boldface.

 
For the three-dimensional structure comparison of the eight available structures, we used the program TOP (18) with the new option MAPS, allowing multiple alignments of protein structures . In addition, the program produces two ranking scores: the sequence identities of aligned residues and the structural diversity . The structural-diversity score was defined as RMS/(Nmatch/N0)3/2, where RMS is the root mean square deviation of the distances between matched C{alpha} atoms, Nmatch is the number of matching residues, and (Nmatch/N0) is the matching fraction of two compared structures . N0 = (N1 + N2/2), where N1 and N2 are the numbers of amino acids in the two compared proteins . This score estimates the evolutionary distance between proteins . These two scores are shown in Table 1 for all known X-ray structures .


TABLE 1 . Sequence identities of aligned residues and structure diversity among proteins

 
Figure 1 displays the proposed alignment and numbering . Interestingly, the numbering of the important class B residues is conserved between old and new alignments . Improvements in the alignment concern mainly N and C termini and small shifts along the sequences . The main result of the new alignment is the identification of 14 sequence fragments of structurally conserved positions, which cover the entirety of all sequences (Fig . 1); they belong mainly to secondary-structure elements ({alpha} helices or ß sheets) . Notably, all Zn ligands are structurally aligned .

The following comments can be made . (i) Only sequences of proteins of known structures are shown . (ii) For residues in lightface, the fact that they have the same number does not imply that they are structurally equivalent . (iii) For newly discovered enzymes, any insertion departing from the present numbering can be characterized by lowercase letters following the number of the last residue of the consensus sequence .

Table 2 shows the numbering of the putative zinc ligands . Not all proteins of known sequence are shown . Only enzymes with <50% sequence identity compared to the first reported sequence are included in the table .


TABLE 2 . Numbering of important class B residues

 
In 1997, Neuwald et al . (23) detected a few proteins that have sequence similarities to (and may have given rise to) Zn-ß-lactamases . They include enzymes with large variations in function (sulfatase; DNA cross-link repair enzyme) and which are encoded by yeast, plant, or bacterial open reading frames . Human glyoxalase II was also shown to belong to the superfamily . More recently, 17 groups with known functions were identified (9) . In order to evaluate the structural diversity of the Zn-ß-lactamase superfamily, human glyoxalase II (3) and rubredoxin oxygen-oxidoreductase from Desulfovibrio gigas (12) were also aligned using TOP, along with one member of each subclass . Table 3 shows the sequence identities and structural diversity of the two proteins and BCII, CphA, and FEZ-1 . As expected, low sequence identity corresponds to a high structural-diversity score . The structural-diversity scores for proteins belonging to a superfamily range from 1.4 to 2, in contrast to 3.5 to 4 for proteins with different folds (18) . Interestingly and surprisingly, FEZ-1 is closer to glyoxalase II and rubredoxin oxygen-oxidoreductase than to BcII or CphA .


TABLE 3 . Sequence identities of aligned residues and structural diversity among proteins

 
In the structural alignment, a large number of amino acid changes and insertions-deletions are observed . One hypothesis is that an ancient protein gave rise to the different subclasses of Zn-ß-lactamases . A few candidates for the ancient protein are those related to essential biological functions within the cell, such as DNA or RNA processing or DNA repair (9) . Nature used a limited number of scaffolds to generate a large variety of biological functions . Zn-ß-lactamases are good examples of such a selection .

 


   ACKNOWLEDGMENTS

 
This work was supported by a grant from the European Union (HPRN-CT-2002-00264) and PAI P5/33 from the Belgian government .


   FOOTNOTES

 
* Corresponding author . Mailing address: Institut de Biologie Structurale "Jean-Pierre Ebel," CEA-CNRS-UJF, Laboratoire de Cristallographie Macromoléculaire, 41 rue Jules Horowitz, F-38027 Grenoble, France . Phone: 33 438 78 56 09 . Fax: 33 438 78 54 94 . E-mail: otto{at}ibs.fr .

The views expressed in this Commentary do not necessarily reflect the views of the journal or of ASM.


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