Lactonases from Bacillus species hydrolyze the N-acylhomoserine lactone (AHL) signaling molecules used in quorum-sensing pathways of many Gram-negative bacteria, including Pseudomonas aeruginosa and Erwinia carotovora, both significant pathogens. Because of sequence similarity, these AHL lactonases have been assigned to the metallo-beta-lactamase superfamily of proteins, which includes metalloenzymes of diverse activity, mechanism, and metal content. However, a recent study claims that AHL lactonase from Bacillus sp. 240B1 is not a metalloprotein [Wang, L. H., et al. (2004) J. Biol. Chem. 279, 13645]. Here, the gene for an AHL lactonase from Bacillus thuringiensis is cloned, and the protein is expressed, purified, and found to bind 2 equiv of zinc. The metal-bound form of AHL lactonase catalyzes the hydrolysis of N-hexanoyl-(S)-homoserine lactone but not the (R) enantiomer. Removal of both zinc ions results in loss of activity, and reconstitution with zinc restores activity, indicating the importance of metal ions for catalytic activity. Metal content, sequence alignments, and X-ray absorption spectroscopy of the zinc-containing lactonase all support a proposed dinuclear zinc binding site similar to that found in glyoxalase II.

Metallo-␤-lactamases (M␤Ls) are zinc-dependent enzymes able to hydrolyze and inactivate most ␤-lactam antibiotics. The large diversity of active site structures and metal content among M␤Ls from different sources has limited the design of a pan-M␤L inhibitor. Here we report the biochemical and biophysical characterization of a novel M␤L, GOB-18, from a clinical isolate of a Gram-negative opportunistic pathogen, Elizabethkingia meningoseptica. Different spectroscopic techniques, three-dimensional modeling, and mutagenesis experiments, reveal that the Zn(II) ion is bound to Asp 120 , His 121 , His 263 , and a solvent molecule, i.e. in the canonical Zn2 site of dinuclear M␤Ls. Contrasting all other related M␤Ls, GOB-18 is fully active against a broad range of ␤-lactam substrates using a single Zn(II) ion in this site. These data further enlarge the structural diversity of M␤Ls.The expression of ␤-lactam degrading enzymes (␤-lactamases) is the most common mechanism of antibiotic resistance among bacteria (1, 2). These enzymes have been grouped into four classes (A-D) according to sequence homology (3, 4). Class A, C, and D enzymes use an active site serine residue as a nucleophile, whereas class B lactamases (generically termed metallo-␤-lactamases, M␤Ls) 9 employ one or two Zn(II) ions to cleave the ␤-lactam ring.M␤Ls have particular importance in the clinical setting since they can hydrolyze a broader spectrum of ␤-lactam substrates than the serine-type enzymes and are resistant to most clinically employed inhibitors (5-11). The design of an efficient pan-M␤L inhibitor has been mostly limited by a striking diversity in the active site structures, catalytic features, and metal ion requirements for activity among different enzymes. Based on this heterogeneity, M␤Ls have been classified into three subclasses: B1, B2, and B3 (3, 6). Subclass B1 includes several chromosomally encoded enzymes such as BcII from Bacillus cereus (12-14), CcrA from Bacteroides fragilis (15-18), BlaB from Elizabethkingia meningoseptica (formerly, Chryseobacterium meningosepticum) (19), as well as the transferable VIM (20)-, IMP (21, 22)-, SPM (23, 24)-, and GIM-type enzymes. Subclass B2 includes the CphA (25, 26) and ImiS (27) lactamases from Aeromonas species. Subclass B3, originally represented only by L1 from Stenotrophomonas maltophilia (28 -30), now includes enzymes from other opportunistic pathogens like FEZ-1 from Legionella gormanii (31) and GOB from E. meningoseptica (32), as well as from environmental bacteria such as CAU-1 from Caulobacter crescentus (33) and THIN-B from Janthinobacterium lividum (34).Molecular structures of M␤Ls from the three subclasses have been solved by x-ray crystallography (12,14,15,25,31). Comparison of the tertiary structure of enzymes belonging to the different subclasses reveals a common ␣␤/␤␣ sandwich fold, in which different insertions and deletions have resulted in different loop topologies and, ultimately, in different zinc coordination environments and metal site occupancies among B1, B2, and B3 en...

The enzyme dimethylargininase (also known as dimethylarginine dimethylaminohydrolase or DDAH; EC 3.5.3.18) catalyzes the hydrolysis of endogenous nitric oxide synthase inhibitors, N(omega)-methyl-l-arginine and N(omega),N(omega)-dimethyl-l-arginine. Understanding the mechanism and regulation of DDAH activity is important for developing ways to control nitric oxide production during angiogenesis and in many cases of vascular endothelial pathobiology. Several possible physiological regulation mechanisms of DDAH depend upon the presence of an active-site cysteine residue, Cys249 in Pseudomonas aeruginosa (Pa) DDAH, which is proposed to serve as a nucleophile in the catalytic mechanism. Through the use of pH-dependent ultraviolet and visible (UV-vis) difference spectroscopy and inactivation kinetics, the pK(a) of the active-site Cys249 in the resting enzyme was found to be unperturbed from pK(a) values of typical noncatalytic cysteine residues. In contrast, the pH dependence of k(cat) values indicates a much lower apparent pK(a) value. UV-vis difference spectroscopy between wild-type and C249S DDAH shows absorbance changes consistent with Cys249 deprotonation to the anionic thiolate upon binding positively charged ligands. The proton from Cys249 is lost either to the solvent or to an unidentified general base. A mutation of the active-site histidine residue, H162G, does not eliminate cysteine nucleophilicity, further arguing against a pre-formed ion pair with Cys249. Finally, UV-vis and X-ray absorption spectroscopy revealed that inhibitory metal ions can bind at these two active-site residues, Cys249 and His162, and also stabilize the anionic form of Cys249. These results support a proposed substrate-assisted mechanism for Pa DDAH in which ligand binding modulates the reactivity of the active-site cysteine.

In an effort to probe Co(II) binding to metallo-beta-lactamase CcrA, EPR, EXAFS, and (1)H NMR studies were conducted on CcrA containing 1 equiv (1-Co(II)-CcrA) and 2 equiv (Co(II)Co(II)-CcrA) of Co(II). The EPR spectra of 1-Co(II)-CcrA and Co(II)Co(II)-CcrA are distinct and indicate 5/6-coordinate Co(II) ions. The EPR spectra also reveal the absence of significant spin-exchange coupling between the Co(II) ions in Co(II)Co(II)-CcrA. EXAFS spectra of 1-Co(II)-CcrA suggest 5/6-coordinate Co(II) with two or more histidine ligands. EXAFS spectra of Co(II)Co(II)-CcrA also indicate 5/6 ligands at a similar average distance to 1-Co(II)-CcrA, including an average of about two histidines per Co(II). (1)H NMR spectra for 1-Co(II)-CcrA revealed seven paramagnetically shifted resonances, three of which were solvent-exchangeable, while the NMR spectra for Co(II)Co(II)-CcrA showed at least 16 shifted resonances, including an additional solvent-exchangeable resonance and a resonance at 208 ppm. The data indicate sequential binding of Co(II) to CcrA and that the first Co(II) binds to the consensus Zn(1) site in the enzyme.

In an effort to probe the structure, mechanism, and biochemical properties of metallo-β-lactamase Bla2 from Bacillus anthracis, the enzyme was over-expressed, purified, and characterized. Metal analyses demonstrated that recombinant Bla2 tightly binds 1 eq of Zn(II). Steady-state kinetic studies showed that mono-Zn(II) Bla2 (1Zn-Bla2) is active, while di-Zn(II) Bla2 (ZnZn-Bla2) was unstable. Catalytically, 1Zn-Bla2 behaves like the related enzymes CcrA and L1. In contrast, diCo(II) Bla2 (CoCo-Bla2) is substantially more active than the mono-Co(II) analog. Rapid kinetics and UV-Vis, 1 H NMR, EPR, and EXAFS spectroscopic studies show that Co(II) binding to Bla2 is distrubuted, while EXAFS shows that Zn(II) binding is sequential. To our knowledge, this is the first documented example of a Zn enzyme that binds Co(II) and Zn(II) via distinct mechanisms, underscoring the need to demonstrate transferability when extrapolating results on Co(II)-substituted proteins to the native Zn(II)-containing forms.

Extended X-ray absorption fine structure studies of the metallo-beta-lactamase L1 from Stenotrophomonas maltophilia containing 1 and 2 equiv of Zn(II) and containing 2 equiv of Zn(II) plus hydrolyzed nitrocefin are presented. The data indicate that the first, catalytically dominant metal ion is bound by L1 at the consensus Zn1 site. The data further suggest that binding of the first metal helps preorganize the ligands for binding of the second metal ion. The di-Zn enzyme displays a well-defined metal-metal interaction at 3.42 A. Reaction with the beta-lactam antibiotic nitrocefin results in a product-bound species, in which the ring-opened lactam rotates in the active site to present the S1 sulfur atom of nitrocefin to one of the metal ions for coordination. The product bridges the two metal ions, with a concomitant lengthening of the Zn-Zn interaction to 3.62 A.

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X-ray absorption spectroscopy was used to investigate the metal-binding sites of ImiS from Aeromonas Veronii bV. sobria in catalytically active (1-Zn), product-inhibited (1-Zn plus imipenem), and inactive (2-Zn) forms. The first equivalent of zinc(II) was found to bind to the consensus Zn 2 site. The reaction of 1-Zn ImiS with imipenem leads to a product-bound species, coordinated to Zn via a carboxylate group. The inhibitory binding site of ImiS was examined by a comparison of wild-type ImiS with 1 and 2 equiv of bound zinc. 2-Zn ImiS extended X-ray absorption fine structure data support a binding site that is distant from the active site and contains both one sulfur donor and one histidine ligand. On the basis of the amino acid sequence of ImiS and the crystal structure of CphA [Garau et al. (2005) J. Mol. Biol. 345,[785][786][787][788][789][790][791][792][793][794][795], we propose that the inhibitory binding site is formed by M146, found on the B2-distinct R3 helix, and H118, a canonical Zn 1 ligand, proposed to help activate the nucleophilic water. The mutation of M146 to isoleucine abolishes metal inhibition. This is the first characterization of ImiS with the native metal Zn and establishes, for the first time, the location of the inhibitory metal site.