High-Resolution Crystal Structure of the Subclass B3 Metallo-β-Lactamase BJP-1: Rational Basis for Substrate Specificity and Interaction with Sulfonamides (original) (raw)
Related papers
Antimicrobial Agents and Chemotherapy, 2010
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.
Journal of Molecular Biology, 2008
One mechanism by which bacteria can escape the action of β-lactam antibiotics is the production of metallo-β-lactamases. Inhibition of these enzymes should restore the action of these widely used antibiotics. The tetrameric enzyme L1 from Stenotrophomonas maltophilia was used as a model system to determine a series of high-resolution crystal structures of apo, mono and bi-metal substituted proteins as well as protein-inhibitor complexes. Unexpectedly, although the apo structure revealed only few significant structural differences from the holo structure, some inhibitors were shown to induce amino acid side-chain rotations in the tightly packed active site. Moreover, one inhibitor employs a new binding mode in order to interact with the di-zinc center. This structural information could prove essential in the process of elucidation of the mode of interaction between a putative lead compound and metallo-β-lactamases, one of the main steps in structure-based drug design.
Frontiers in Chemistry
β-Lactams are the most widely employed antibiotics in clinical settings due to their broad efficacy and low toxicity. However, since their first use in the 1940s, resistance to β-lactams has proliferated to the point where multi-drug resistant organisms are now one of the greatest threats to global human health. Many bacteria use β-lactamases to inactivate this class of antibiotics via hydrolysis. Although nucleophilic serine-β-lactamases have long been clinically important, most broad-spectrum β-lactamases employ one or two metal ions (likely Zn2+) in catalysis. To date, potent and clinically useful inhibitors of these metallo-β-lactamases (MBLs) have not been available, exacerbating their negative impact on healthcare. MBLs are categorised into three subgroups: B1, B2, and B3 MBLs, depending on their sequence similarities, active site structures, interactions with metal ions, and substrate preferences. The majority of MBLs associated with the spread of antibiotic resistance belong...
PLoS Pathogens, 2014
Pseudomonas aeruginosa is one of the most virulent and resistant non-fermenting Gram-negative pathogens in the clinic. Unfortunately, P. aeruginosa has acquired genes encoding metallo-b-lactamases (MbLs), enzymes able to hydrolyze most blactam antibiotics. SPM-1 is an MbL produced only by P. aeruginosa, while other MbLs are found in different bacteria. Despite similar active sites, the resistance profile of MbLs towards b-lactams changes from one enzyme to the other. SPM-1 is unique among pathogen-associated MbLs in that it contains ''atypical'' second sphere residues (S84, G121). Codon randomization on these positions and further selection of resistance-conferring mutants was performed. MICs, periplasmic enzymatic activity, Zn(II) requirements, and protein stability was assessed. Our results indicated that identity of second sphere residues modulates the substrate preferences and the resistance profile of SPM-1 expressed in P. aeruginosa. The second sphere residues found in wild type SPM-1 give rise to a substrate selectivity that is observed only in the periplasmic environment. These residues also allow SPM-1 to confer resistance in P. aeruginosa under Zn(II)-limiting conditions, such as those expected under infection. By optimizing the catalytic efficiency towards b-lactam antibiotics, the enzyme stability and the Zn(II) binding features, molecular evolution meets the specific needs of a pathogenic bacterial host by means of substitutions outside the active site.
Protein Science, 2008
The metallo-P-lactamases require zinc or cadmium for hydrolyzing p-lactam antibiotics and are inhibited by mercurial compounds. To date, there are no clinically useful inhibitors of this class of enzymes. The crystal structure of the Zn2+-bound enzyme from Bacteroides fragilis contains a binuclear zinc center in the active site. A hydroxide, coordinated to both zinc atoms, is proposed as the moiety that mounts the nucleophilic attack on the carbonyl carbon atom of the p-lactam ring. To study the metal coordination further, the crystal structures of a Cd*+-bound enzyme and of an Hg'+-soaked zinc-containing enzyme have been determined at 2. I A and 2.7 A, respectively. Given the diffraction resolution, the Cd2+-bound enzyme exhibits the same active-site architecture as that of the Zn2+-bound enzyme, consistent with the fact that both forms are enzymatically active. The IO-fold reduction in activity of the Cd2+-bound molecule compared with the Zn'+-bound enzyme is attributed to fine differences in the charge distribution due to the difference in the ionic radii of the two metals. In contrast, in the Hg2+-bound structure, one of the zinc ions, Zn2, was ejected, and the other zinc ion, Zn I , remained in the same site as in the 2-Zn2+-bound structure. Instead of the ejected zinc, a mercury ion binds between Cys 104 and Cys 181, 4.8 A away from Znl and 3.9 A away from the site where Zn2 is located in the 2-Zn2+-bound molecule. The perturbed binuclear metal cluster explains the inactivation of the enzyme by mercury compounds.
Crystal structure of the wide-spectrum binuclear zinc β-lactamase from Bacteroides fragilis
Structure, 1996
The metallo--lactamase from Bacteroides fragilis hydrolyzes a wide range of -lactam antibiotics, and is not clinically susceptible to any known -lactamase inhibitors. B. fragilis is associated with post-surgery hospital infections, and there has been a recent report of plasmid-mediated dissemination of the enzyme. Effective inhibitors are therefore urgently needed. Knowledge of the three-dimensional structure will aid in the drug design effort.
Journal of molecular biology, 2011
Metallo-β-lactamases (MBLs) or class B β-lactamases are zinc-dependent enzymes capable of inactivating almost all classes of β-lactam antibiotics. To date, no MBL inhibitors are available for clinical use. Of the three MBL subclasses, B2 enzymes, unlike those from subclasses B1 and B3, are fully active with one zinc ion bound and possess a narrow spectrum of activity, hydrolyzing carbapenem substrates almost exclusively. These remain the least studied MBLs. Sfh-I, originally identified from the aquatic bacterium Serratia fonticola UTAD54, is a divergent member of this group. Previous B2 MBL structures, available only for the CphA enzyme from Aeromonas hydrophila, all contain small molecules bound in their active sites. In consequence, the mechanism by which these enzymes activate the water nucleophile required for β-lactam hydrolysis remains to be unambiguously established. Here we report crystal structures of Sfh-I as a complex with glycerol and in the unliganded form, revealing for the first time the disposition of water molecules in the B2 MBL active site. Our data indicate that the hydrolytic water molecule is activated by His118 rather than by Asp120 and/or zinc. Consistent with this proposal, we show that the environment of His118 in B2 MBLs is distinct from that of the B1 and B3 enzymes, where this residue acts as a zinc ligand, and offer a structure-based mechanism for β-lactam hydrolysis by these enzymes.► We present two crystal structures of the mono-zinc MBL Sfh-I. ► The first structures to locate water molecules in the mono-zinc MBL active site. ► His118, rather than Asp120 or zinc, activates the water nucleophile.
Antimicrobial agents and chemotherapy, 2016
Metallo-beta-lactamases (MBLs) are broad spectrum, Zn(II) dependent lactamases able to confer resistance to virtually every β-lactam antibiotic currently available. The large diversity of active site structures and metal content among MBLs from different sources has limited the design of a pan-MBL inhibitor. GOB-18 is a divergent MBL from subclass B3, expressed by the opportunistic Gram-negative pathogen Elizabethkingia meningoseptica This MBL is atypical since several residues conserved in B3 enzymes (such as a metal ligand His) are substituted in GOB enzymes. Here we report the crystal structure of the periplasmic di-Zn(II) form of GOB-18. This enzyme displays a unique active site structure, with residue Gln116 coordinating the Zn1 ion through its terminal amide moiety, replacing a ubiquitous His residue. This situation contrasts with that of B2 MBLs, where an equivalent His116Asn substitution leads to a di-Zn(II) inactive species. Instead, both the mono- and di-Zn(II) forms of GO...
Biochemistry, 2000
Metallo -lactamase enzymes confer antibiotic resistance to bacteria by catalyzing the hydrolysis of -lactam antibiotics. This relatively new form of resistance is spreading unchallenged as there is a current lack of potent and selective inhibitors of metallo -lactamases. Reported here are the crystal structures of the native IMP-1 metallo -lactamase from Pseudomonas aeruginosa and its complex with a mercaptocarboxylate inhibitor, 2-[5-(1-tetrazolylmethyl)thien-3-yl]-N-[2-(mercaptomethyl)-4-(phenylbutyrylglycine)]. The structures were determined by molecular replacement, and refined to 3.1 Å (native) and 2.0 Å (complex) resolution. Binding of the inhibitor in the active site induces a conformational change that results in closing of the flap and transforms the active site groove into a tunnel-shaped cavity enclosing 83% of the solvent accessible surface area of the inhibitor. The inhibitor binds in the active site through interactions with residues that are conserved among metallo -lactamases; the inhibitor's carboxylate group interacts with Lys161, and the main chain amide nitrogen of Asn167. In the "oxyanion hole", the amide carbonyl oxygen of the inhibitor interacts through a water molecule with the side chain of Asn167, the inhibitor's thiolate bridges the two Zn(II) ions in the active site displacing the bridging water, and the phenylbutyryl side chain binds in a hydrophobic pocket (S1) at the base of the flap. The flap is displaced 2.9 Å compared to the unbound structure, allowing Trp28 to interact edge-to-face with the inhibitor's thiophene ring. The similarities between this inhibitor and the -lactam substrates suggest a mode of substrate binding and the role of the conserved residues in the active site. It appears that the metallo -lactamases bind their substrates by establishing a subset of binding interactions near the catalytic center with conserved characteristic chemical groups of the -lactam substrates. These interactions are complemented by additional nonspecific binding between the more variable groups in the substrates and the flexible flap. This unique mode of binding of the mercaptocarboxylate inhibitor in the enzyme active site provides a binding model for metallo -lactamase inhibition with utility for future drug design.
A minimalistic approach to identify substrate binding features in B1 Metallo-[beta]-lactamases
Bioorganic & medicinal …, 2007
The 2-oxoazetidinylacetate sodium salt was synthesized as a model of a minimal b-lactam drug. This compound and the monobactam aztreonam were assayed as substrates of the Metallo-b-lactamase BcII. None of them was hydrolyzed by the enzyme. While the azetidinone was not able to bind BcII, aztreonam was shown to bind in a nonproductive mode. These results provide an explanation for the unability of Metallo-b-lactamases to inactive monobactams and give some clues for inhibitor design.