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...

Metallo-β-lactamases (MBLs) hydrolyze almost all β-lactam antibiotics and are unaffected by clinically available β-lactamase inhibitors (βLIs). Active-site architecture divides MBLs into three classes (B1, B2, and B3), complicating development of βLIs effective against all enzymes. Bisthiazolidines (BTZs) are carboxylate-containing, bicyclic compounds, considered as penicillin analogs with an additional free thiol. Here, we show both L-and D-BTZ enantiomers are micromolar competitive βLIs of all MBL classes in vitro, with K i s of 6-15 μM or 36-84 μM for subclass B1 MBLs (IMP-1 and BcII, respectively), and 10-12 μM for the B3 enzyme L1. Against the B2 MBL Sfh-I, the L-BTZ enantiomers exhibit 100-fold lower K i s (0.26-0.36 μM) than D-BTZs (26-29 μM). Importantly, cell-based time-kill assays show BTZs restore β-lactam susceptibility of Escherichia coli-producing MBLs (IMP-1, Sfh-1, BcII, and GOB-18) and, significantly, an extensively drug-resistant Stenotrophomonas maltophilia clinical isolate expressing L1. BTZs therefore inhibit the full range of MBLs and potentiate β-lactam activity against producer pathogens. X-ray crystal structures reveal insights into diverse BTZ binding modes, varying with orientation of the carboxylate and thiol moieties. BTZs bind the di-zinc centers of B1 (IMP-1; BcII) and B3 (L1) MBLs via the free thiol, but orient differently depending upon stereochemistry. In contrast, the L-BTZ carboxylate dominates interactions with the monozinc B2 MBL Sfh-I, with the thiol uninvolved. D-BTZ complexes most closely resemble β-lactam binding to B1 MBLs, but feature an unprecedented disruption of the D120-zinc interaction. Cross-class MBL inhibition therefore arises from the unexpected versatility of BTZ binding.carbapenemase | antibiotic resistance | inhibitors | bisthiazolidines | metallo-β-lactamase

Keywords:Glycosyl hydrolase family 30 (GH30) Glycosyl hydrolase family 5 (GH5) Glucuronoxylan xylanohydrolase Endo-b-1,6-galactanase Glucosylceramidase Endo-b-1,6-glucanase a b s t r a c tIn this work glycosyl hydrolase (GH) family 30 (GH30) is analyzed and shown to consist of its currently classified member sequences as well as several homologous sequence groups currently assigned within family GH5. A large scale amino acid sequence alignment and a phylogenetic tree were generated and GH30 groups and subgroups were designated. A partial rearrangement in the GH30 defining side-associated b-domain contributes to the differentiation of two major groups that contain up to eight subgroups. For this CAZy family of Clan A enzymes the dual domain fold is conserved, suggesting that it may be a requirement for evolved function. This work redefines GH family 30 and serves as a guide for future efforts regarding enzymes classified within this family.Published by Elsevier B.V. on behalf of the Federation of European Biochemical Societies.

Recent studies have shown that liver cirrhosis (LC) behaves as an acquired hypercoagulable state with increased thrombotic risk. This is why anticoagulation therapy (AT) is now frequently used in these patients. Variceal bleeding is a severe complication of LC. It is unknown whether AT may impact the outcome of bleeding in these patients. Fifty-two patients on AT with upper gastrointestinal bleeding (UGIB) were evaluated. Portal vein thrombosis (PVT) and different cardiovascular disorders (CVDs) were the indication for AT in 14 and 38 patients, respectively. Overall, 104 patients with LC and UGIB not under AT matched for severity of LC, age, sex, source of bleeding, and Sequential Organ Failure Assessment (SOFA) score served as controls. UGIB was attributed to portal hypertension (PH) in 99 (63%) patients and peptic/vascular lesions in 57 (37%). Twenty-six (17%) patients experienced 5-day failure; SOFA, source of UGIB, and PVT, but not AT, were independent predictors of 5-day failure. In addition, independent predictors of 6-week mortality, which was observed in 26 (11%) patients, were SOFA, Charlson Comorbidity index, and use of AT for a CVD. There were no differences between patients with/without AT in needs for rescue therapies, intensive care unit admission, transfusions, and hospital stay. Conclusions: Factors that impact the outcome of UGIB in patients under AT are degree of multiorgan failure and comorbidity, but not AT itself. (HEPATOLOGY 2015;62:575-583) T he use of anticoagulant therapy (AT) for prevention and management of thrombotic events may complicate the management of upper gastrointestinal bleeding (UGIB), increasing the morbidity/mortality associated to it. [1][2][3][4] Until recently, liver cirrhosis (LC) has been considered a hypocoagulant and prohemorrhagic condition owing reduction in platelet count and increase in prothrombin time. Currently, this paradigm has been challenged by the observation that patients with