Standard Numbering Scheme for Class B β-Lactamases

Metallo-β-lactamases constitute the molecular class B of Ambler (1) and group 3 according to the Bush-Jacoby-Medeiros functional classification (6). In recent years, many new enzymes of this class have been described and the sequences of the corresponding genes have been determined. Their clinical importance is highlighted by the fact that they hydrolyze carbapenems, compounds which most often escape the activity of active-site serine β-lactamases. Moreover, most metallo-β-lactamases are broad-spectrum enzymes which also hydrolyze a variety of penicillins and cephalosporins (13, 21, 22, 26). On the basis of the known sequences, three different lineages, identified as subclasses B1, B2, and B3, can be characterized. Subclass B1 contains most known metallo-β-lactamases, including the β-lactamase II (BcII) proteins from Bacillus cereus or other Bacillus spp. (15, 16, 19) and Bacillus sp. strain 170 (16), the CcrA (24) (also named CfiA [29]) proteins of Bacteroides fragilis, the BlaB proteins from Chryseobacterium meningosepticum (2, 26, 34), the IND-1 enzyme from Chryseobacterium indologenes (3), the IMP proteins found in some clinical isolates of Pseudomonas aeruginosa (17, 28), Serratia marcescens (21), Klebsiella pneumoniae (GenBank EMBL accession no. D29636), and Acinetobacter baumannii (25), and the VIM proteins found in some P. aeruginosa clinical isolates (18, 22). Subclass B2 includes the enzymes produced by various species of Aeromonas (CphA [20], ImiS [33], and CphA2 [23]) and the Sfh-I β-lactamase (GenBank accession no. AF197943) from Serratia fonticola. Finally, subclass B3 includes the L1 proteins from Stenotrophomonas maltophilia (27, 32), the GOB proteins from C. meningosepticum (2), the FEZ-1 enzyme from Legionella gormanii (5), and the THIN-B β-lactamase produced by Janthinobacterium lividum (25a).

The three-dimensional structures of several B1 (BcII [7, 9, 12], CcrA [8, 10], and IMP-1 [11]) enzymes and one B3 (L1 [31]) enzyme have been solved by X-ray crystallography. Despite a very low degree of sequence similarity between the two subclasses, the general structures and the relative positions of the secondary structure elements are similar. Surprisingly, the L1 enzyme is a tetramer (4, 31), whereas the B1, B2, and other B3 (FEZ-1 [5; P. S. Mercuri, F. Bouillenne, L. Boschi, J. Lamotte-Brasseur, G. Amicosante, B. Devreese, J. van Beeumen, J. M. Frère, G. M. Rossolini, and M. Galleni, unpublished data] and GOB-1 [2]) β-lactamases so far studied are monomers.

There are, however, no doubts that the proteins are homologous and the sequences of representatives of the three subclasses can be easily aligned. Indeed, in addition to the expected differences at the N and C termini, several insertions and deletions are necessary to allow the alignment of the few conserved residues acting, for instance, as ligands of the two zinc ions which can bind at the active site. Thus, homologous residues from the different class B sequences which are known to play a relevant role in the structure and function often differ in their numbering, even within each subclass.

In order to facilitate the comparative analysis of the structures and of the catalytic mechanisms, we would like to propose a standard numbering scheme for the class B β-lactamases, the BBL numbering, by analogy with the ABL numbering which has been widely accepted for class A β-lactamases. For the class B enzymes, the task was complicated by insertions and deletions and by the generally low degree of similarity but facilitated by the availability of some three-dimensional structures, which allowed the identification of homologous secondary structure elements, even when the sequence similarity was not obvious.

Figure 1 shows the proposed alignment and the derived numbering. The observed (B1 and B3) and expected (B2) secondary structure elements are indicated.

FIG. 1.

Alignment of 12 class B β-lactamases numbered according to the BBL scheme. The sequences are referred to by their familiar names. BcII, Bacillus cereus 569H (15); IMP-1, Pseudomonas aeruginosa 101/477 (17); CcrA, Bacteroides fragilis TAL3636 (24); VIM-1, Pseudomonas aeruginosa VR-143/97 (18); BlaB, Chryseobacterium meningosepticum NCTC10585 (26); IND-1, Chryseobacterium indologenes 001 (3); CphA, Aeromonas hydrophila AE036 (20); Sfh-I, Serratia fonticola UTAD54 (GenBank accession no. AF197943); L1, Stenotrophomonas maltophilia IID1275 (32); FEZ-1, Legionella gormanii ATCC33297^T (5); GOB-1, Chryseobacterium meningosepticum PINT (2); and THIN-B, Janthinobacterium lividum JAC1 (25a). The names written in bold refer to the enzymes for which the three-dimensional structure is known. The amino acid in bold (Ala 22 of L1) represents the first amino acid of the mature β-lactamase. Conserved secondary structure elements of subclasses B1 and B3 are indicated above the sequences: 3₁₀, 3₁₀ helix; S, β strand; H, helix. Secondary structure elements specific to subclasses B1 and B3 are highlighted by italic characters above and under the sequences, respectively. Amino acid insertions in newly sequenced enzymes are represented by small letters. The residues acting as zinc ligands in at least one subclass are characterized as follows: z, conserved residues in the three subclasses; ·, conserved residues in subclass B1 and some enzymes of subclass B3; +, conserved residue in subclass B3; §, conserved residues in subclasses B1 and B2.

The following comments can be made. (i) Not all the known sequences are shown. When variants of an enzyme are known and the amino acid alignment exhibits more than 80% sequence identity, only the first described sequence is included in the alignment.

(ii) Alignments at the N and C termini are rather uncertain, due to a high variability even within each subclass. As is done for the class A enzymes, residue no. 1 is the first residue of the leader peptide sequence of the S. maltophilia L1 protein (32). Since they are highly divergent and irrelevant to the functional structure, the other leader sequences have not been included unless the site of action of the signal peptidase has not been verified (Sfh-I [GenBank accession no. AF197943], IND-1 [3], and THIN-B [25a]).

(iii) This is only a numbering scheme. The fact that residues in different proteins have been assigned the same number does not imply that they occupy exactly the same relative spatial position. Indeed, if the Zn ions and their ligands are superimposed, the G₂₃₂N₂₃₃ dyad of BcII is more than 3 Å away from the corresponding residues in the S. maltophilia enzyme.

(iv) The loop which can close the active site of B1 enzymes extends between residues BBL 61 and 65 (11, 14, 30). It is absent in subclass B3 (31) and probably in B2.

(v) Any insert in a newly discovered enzyme can be characterized by small letters following the number of the last residue of the consensus sequence. Accordingly, residues N₁₄₀G₁₄₁ of THIN-B are defined as BBL 150a and -b and residues I₁₉₈EQG₂₀₁ of Sfh-I are defined as BBL 252a, -b, -c and -d, respectively.

(vi) Table 1 shows a cross-reference of the BBL numbering of the residues identified as or suspected to be the Zn1 and Zn2 ligands and that used for the individual enzymes up to the present time. Note that in subgroup B3, one of the Zn2 ligands (H121) originates with a very different part of the polypeptide chain compared to subgroup B1. Similarly, in subclass B2 and for the B3 GOB-1 enzyme, the sequence alignments unambiguously point to residues H118, H196, and N116 (B2) or Q116 (B3), but such a function is rather unusual for asparagine and glutamine side chains.

TABLE 1.

Numbering of the important class B residues^a

β-Lactamase	Zn1 ligands			Zn2 ligands
Subclass B1
Consensus BBL	His116	His118	His196	Asp120	Cys221	His263
BcII	His86	His88	His149	Asp90	Cys168	His210
IMP-1	His77	His79	His139	Asp81	Cys158	His197
CcrA	His99	His101	His162	Asp 103	Cys181	His223
VIM-1	His88	His90	His153	Asp92	Cys172	His214
BlaB	His76	His78	His139	Asp80	Cys158	His200
IND-1	His96	His98	His159	Asp100	Cys178	His220
Subclass B2
Consensus BBL	Asn116	His118	His196	Asp120	Cys221	His263
CphA	Asn69	His71	His148	Asp73	Cys167	His205
Sfh-I	Asn72	His74	His151	Asp76	Cys170	His212
Subclass B3
Consensus BBL	His/Gln116	His118	His196	Asp120	His121	His263
L1	His84	His86	His160	Asp88	His89	His225
FEZ-1	His71	His73	His149	Asp75	His76	His215
GOB-1	Gln80	His82	His157	Asp84	His85	His213
THIN-B	His105	His107	His185	Asp109	His110	His253

^aFor CcrA, the numbering is reported in references 10 and 24. When not confirmed by a three-dimensional structure, the ligands are underlined. The consensus (bold) and putative consensus (bold and underlined) ligand numbers are given for each subgroup. 

Appendix

The metallo-β-lactamase group also includes the following: G. Amicosante and N. Franceschini, Dipartimento di Scienze e Tecnologie Biomediche, Università di L'Aquila, I-67100 Coppito, L'Aquila, Italy; K. Bush, The R. W. Johnson Pharmaceutical Research Institute, Raritan, NJ 08869; N. O. Concha, Department of Structural Biology, SmithKline Beecham Pharmaceuticals, King of Prussia, PA 19406; O. Herzberg, Center for Advanced Research in Biotechnology, University of Maryland Biotechnology Institute, Rockville, MD 20850; D. M. Livermore, Antibiotic Resistance Monitoring and Reference Laboratory, Central Public Health Laboratory, London NW9 5HT, United Kingdom; P. Nordmann, Service de Bactériologie-Virologie, Hopital de Bicêtre, Faculté de Médecine Paris-Sud, 94275 Le Kremlin-Bicêtre, France; B. A. Rasmussen, Wyeth-Ayerst Research, Pearl River, NY 10965; J. Rodrigues and M. J. Saavedra, Department of Animal Health, University of Trás-os-Montes e Alto Douro, 5000-911 Vila Real, Portugal; B. Sutton and S. M. Fabiane, The Randall Centre, King's College London, London SE1 1UL, United Kingdom; and J. H. Toney, Department of Biochemistry, Merck Research Laboratories, Rahway, NJ 07065-0900.