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Antibiotic resistance has emerged as a major threat to global health care. This is largely due to the fact that many pathogens have developed strategies to acquire resistance to antibiotics. Metallo-β-lactamases (MBL) have evolved to inactivate most of the commonly used β-lactam antibiotics. AIM-1 is one of only a few MBLs from the B3 subgroup that is encoded on a mobile genetic element in a major human pathogen. Here, its mechanism of action was characterised with a combination of spectroscopic and kinetic techniques and compared to that of other MBLs. Unlike other MBLs it appears that AIM-1 has two avenues available for the turnover of the substrate nitrocefin, distinguished by the identity of the rate-limiting step. This observation may be relevant with respect to inhibitor design for this group of enzymes as it demonstrates that at least some MBLs are very flexible in terms of interactions with substrates and possibly inhibitors.

Metal ion-dependent, organophosphate-degrading enzymes have acquired increasing attention due to their ability to degrade and thus detoxify commonly used pesticides and nerve agents such as sarin. The best characterized of these enzymes are from Pseudomonas diminuta (OPH) and Agrobacterium radiobacter (OpdA). Despite high sequence homology (>90 % identity) and conserved metal ion coordination these enzymes display considerable variations in substrate specificity, metal ion affinity/preference and reaction mechanism. In this study, we highlight the significance of the presence (OpdA) or absence (OPH) of an extended hydrogen bond network in the active site of these enzymes for the modulation of their catalytic properties. In particular, the second coordination sphere residue in position 254 (Arg in OpdA, His in OPH) is identified as a crucial factor in modulating the substrate preference and binding of these enzymes. Inhibition studies with fluoride also support a mechanism for OpdA whereby the identity of the hydrolysis-initiating nucleophile changes as the pH is altered. The same is not observed for OPH.

BackgroundPresently, enzymes still constitute a major part of the cost of biofuel production from lignocellulosic biomass. Processive endoglucanases, which possess both endoglucanase and exoglucanase activity, have the potential to reduce the costs of biomass saccharification when used together with commercial cellulases. Therefore, the exploration of new processive endoglucanases has attracted much attention with a view to accelerating the industrialization of biofuels and biochemicals.ResultsThe endoglucanase EG5C and its truncated form EG5C-1 from Bacillus subtilis BS-5 were expressed and characterized. EG5C was a typical endoglucanase, comprised of a family 5 catalytic domain and family 3 carbohydrate-binding domain, and which had high activity toward soluble cellulosic substrates, but low activity toward insoluble cellulosic substrates. Importantly, the truncated form EG5C-1 was a processive endoglucanase that hydrolyzed not only carboxymethyl cellulose (CMC), but also insoluble cellulosic substrates. The hydrolytic activities of EG5C-1 towards CMC, phosphoric acid-swollen cellulose (PASC), p-nitrophenyl-β-d-cellobioside, filter paper and Avicel are 4170, 700, 2550, 405 and 320 U/μmol, respectively. These data demonstrated that EG5C-1 had higher activity ratio of exoglucanase to endoglucanase than other known processive endoglucanases. When PASC was degraded by EG5C-1, the ratio of soluble to insoluble reducing sugars was about 3.7 after 3 h of incubation with cellobiose and cellotriose as the main products. Importantly, EG5C-1 alone was able to hydrolyze filter paper and PASC. At 5% substrate concentration and 10 FPU/g PASC enzyme loading, the saccharification yield was 76.5% after 60 h of incubation. Replacement of a phenylalanine residue (F238) by an alanine at the entrance/exit of the substrate binding cleft significantly reduces the ability of EG5C-1 to degrade filter paper and Avicel, but this mutation has little impact on CMCase activity. The processivity of this mutant was also greatly reduced while its cellulose binding ability was markedly enhanced.ConclusionsThe processive endoglucanase EG5C-1 from B. subtilis BS-5 exhibits excellent properties that render it a suitable candidate for use in biofuel and biochemical production from lignocellulosic biomass. In addition, our studies also provide useful information for research on enzyme processivity at the molecular level.Electronic supplementary materialThe online version of this article (10.1186/s13068-018-1022-2) contains supplementary material, which is available to authorized users.

A SAR study on derivatives of 2-amino-1-benzyl-4,5-diphenyl-1H-pyrrole-3-carbonitrile 5a revealed that the 3-carbonitrile group, vicinal 4,5-diphenyl and N-benzyl side chains of the pyrrole are important for the inhibitory potencies of these compounds against members representing the three main subclasses of metallo-β-lactamases (MBLs), i.e. IMP-1 (representing the B1 subgroup), CphA (B2) and AIM-1 (B3). Coupling of 5a with a series of acyl chlorides and anhydrides led to the discovery of two N-acylamide derivatives, 10 and 11, as the two most potent IMP-1 inhibitors in this series. However, these compounds are less effective towards CphA and AIM-1. The N-benzoyl derivative of 5a retained potent in vitro activity against each of MBLs tested (with inhibition constants in the low μM range). Importantly, this compound also significantly enhanced the sensitivity of IMP-1, CphA- or AIM-1-producing cell cultures towards meropenem. This compound presents a promising starting point for the development of a universal MBL inhibitor, targeting members of each of the major subgroups of this family of enzymes.

Broad spectrum antibiotic-degrading metalloβ-lactamases are phylogenetically diverse Dear Editor,

Ketol-acid reductoisomerase (KARI) is a Mg(2+) -dependent enzyme in the branched-chain amino acid biosynthesis pathway. It catalyses a complex two-part reaction: an alkyl migration followed by a NADPH-dependent reduction. Both reactions occur within the one active site, but in particular, the mechanism of the isomerisation step is poorly understood. Here, using a combination of kinetic, thermodynamic and spectroscopic techniques, the reaction mechanisms of both Escherichia coli and rice KARI have been investigated. We propose a conserved mechanism of catalysis, whereby a hydroxide, bridging the two Mg(2+) ions in the active site, initiates the reaction by abstracting a proton from the C2 alcohol group of the substrate. While the μ-hydroxide-bridged dimetallic centre is pre-assembled in the bacterial enzyme, in plant KARI substrate binding leads to a reduction of the metal-metal distance with the concomitant formation of a hydroxide bridge. Only Mg(2+) is capable of promoting the isomerisation reaction, likely to be due to non-competent substrate binding in the presence of other metal ions.

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