High-Resolution Crystal Structure of the Subclass B3 Metallo-β-Lactamase BJP-1: Rational Basis for Substrate Specificity and Interaction with Sulfonamides

Abstract

Metallo-␤-lactamases (MBLs) are important enzymatic factors in resistance to ␤-lactam antibiotics that show important structural and functional heterogeneity. BJP-1 is a subclass B3 MBL determinant produced by Bradyrhizobium japonicum that exhibits interesting properties. BJP-1, like CAU-1 of Caulobacter vibrioides, overall poorly recognizes ␤-lactam substrates and shows an unusual substrate profile compared to other MBLs. In order to understand the structural basis of these properties, the crystal structure of BJP-1 was obtained at 1.4-Å resolution. This revealed significant differences in the conformation and locations of the active-site loops, determining a rather narrow active site and the presence of a unique N-terminal helix bearing Phe-31, whose side chain binds in the active site and represents an obstacle for ␤-lactam substrate binding. In order to probe the potential of sulfonamides (known to inhibit various zinc-dependent enzymes) to bind in the active sites of MBLs, the structure of BJP-1 in complex with 4-nitrobenzenesulfonamide was also obtained (at 1.33-Å resolution), thereby revealing the mode of interaction of these molecules in MBLs. Interestingly, sulfonamide binding resulted in the displacement of the side chain of Phe-31 from its hydrophobic binding pocket, where the benzene ring of the molecule is now found. These data further highlight the structural diversity shown by MBLs but also provide interesting insights in the structure-function relationships of these enzymes. More importantly, we provided the first structural observation of MBL interaction with sulfonamides, which might represent an interesting scaffold for the design of MBL inhibitors.␤-Lactamases are bacterial enzymes that confer resistance to ␤-lactam antibiotics, the most widely used family of anti-infective agents, by hydrolyzing the amide bond of the ␤-lactam ring and thus bear significant clinical relevance (32,41,43). Two structurally and mechanistically distinct families of ␤-lactamases are known: the active-site serine enzymes (Ambler's classes A, C, and D), acting via an acylation-deacylation mechanism, and the metallo-␤-lactamases (MBLs) (Ambler's class B), which require zinc in their active sites and whose catalytic mechanism is less well understood, although several hypotheses have been provided (4, 13, 21, 33). On the basis of sequence homology, three subclasses that also differ by the natures and positions of the residues that constitute the metal binding site(s) have been defined (44).From a structural standpoint, MBLs share a unique fold that was first identified when the structure of the BcII enzyme from Bacillus cereus was solved; this fold consists of a ␤-sandwich flanked by ␣-helices, the metal center being located at the interface of two roughly symmetrical domains (9). Subsequently, many proteins encoded by the genomes of many organisms (from Archaea to mammalians) appeared to share the same typical fold and were grouped in the so-called MBL superfamily, which contains zinc hydrolases that might exhibit a w...

Crystal Structure of the Metallo-β-Lactamase GOB in the Periplasmic Dizinc Form Reveals an Unusual Metal Site

“…As already described for the MBL fold (1-6), the whole domain of GOB-18 can be depicted as a duplication of two structurally similar ␣/␤ hemidomains. GOB-18 is very similar to other B3 lactamases (20)(21)(22)(23)(24), despite low sequence identity. Structural alignment of GOB-18 (chain A) with available B3 lactamase models allows for similarity quantification (Fig.…”

“…All B3 lactamases crystallized so far are characterized by at least one disulfide bridge, in most cases between Cys residues located in the C-terminal helix and the loop linking elements ␣5 and ␤12 (20)(21)(22)(23)(24). GOB-18 is an exception in this regard within B3 enzymes, with only one Cys residue in its primary structure, i.e., Cys180 (201) (in strand ␤10), which is buried within the protein core (Fig.…”

“…Ultimately, Ser221 (equivalent to Cys221 in the DCH site) is no longer a metal ligand, so that the Zn2 site is a DHH site in B3 MBLs. In addition, a third water molecule participates as a fifth ligand, giving rise to a square pyramidal coordination sphere of Zn2 (20)(21)(22)(23)(24). Similar to B1 enzymes, the Zn1 ion in B3 lactamases is regarded as responsible for nucleophile activation (20)(21)(22)(23)(24).…”

“…B3 lactamases, distantly related to the B1 and B2 subclasses, are mostly di-Zn(II) enzymes (20)(21)(22)(23)(24). The Zn1 site of B3 enzymes is a 3-H site similar to that of B1 enzymes, whereas two mutations (Cys221Ser and Arg121His) affect the Zn2 coordination geometry (20)(21)(22)(23)(24), and the metal ion is bonded to Asp120, His121, and His263. Ultimately, Ser221 (equivalent to Cys221 in the DCH site) is no longer a metal ligand, so that the Zn2 site is a DHH site in B3 MBLs.…”

Metallo-β-Lactamases: A Major Threat to Human Health

“…Examples of the B2 and B3 subgroups are CphA from A. hydrophila [21] [22] [67]- [71], ImiS from A. veronii bv. Sobria and Sfh-I from S. fonticola [67] [71] [72], and L1 from S. maltophilia [24] [73]- [75], FEZ-1 from F. gormanii [76], BJP-1 from B. japonicum [77], MIM-1 from N. pentaromativorans [78], MIM-2 from S. agarivorans [78], SMB-1 from S. marcescens [79], CAR-1 from E carotovora [80] and THIN-B from J. lividum [81], while the recently proposed B4 subgroup is represented by SPR-1 from S. proteamaculans [25] and CSA-1 from C. sakazaki [15]. B1-type MBLs have two peptide loops, L3 and L8, in the vicinity of the metal ion-containing active site (Figure 3).These loops are believed to be crucial for the determination of the substrate specificity of these enzymes [2].…”