Kinetic, electrochemical and spectral characterization of bacterial and archaeal rusticyanins; unexpected stability issues and consequences for applications in biotechnology.

Motivated by the ambition to establish an enzyme-driven bioleaching pathway for copper extraction, properties of the Type-1 copper protein rusticyanin from Acidithiobacillus ferrooxidans (AfR) were compared with those from an ancestral form of this enzyme (N0) and an archaeal enzyme identified in Ferroplasma acidiphilum (FaR). While both N0 and FaR show redox potentials similar to that of AfR their electron transport rates were significantly slower. The lack of a correlation between the redox potentials and electron transfer rates indicates that AfR and its associated electron transfer chain evolved to specifically facilitate the efficient conversion of the energy of iron oxidation to ATP formation. In F. acidiphilum this pathway is not as efficient unless it is up-regulated by an as of yet unknown mechanism. In addition, while the electrochemical properties of AfR were consistent with previous data, previously unreported behavior was found leading to a form that is associated with a partially unfolded form of the protein. The cyclic voltammetry (CV) response of AfR immobilized onto an electrode showed limited stability, which may be connected to the presence of the partially unfolded state of this protein. Insights gained in this study may thus inform the engineering of optimized rusticyanin variants for bioleaching processes as well as enzyme-catalyzed solubilization of copper-containing ores such as chalcopyrite.

Structure-guided engineering of branched-chain α-keto acid decarboxylase for improved 1,2,4-butanetriol production by in vitro synthetic enzymatic biosystem.

Efficient synthetic routes for biomanufacturing chemicals often require the overcoming of pathway bottlenecks by tailoring enzymes to improve the catalytic efficiency or even implement non-native activities. 1,2,4-butanetriol (BTO), a valuable commodity chemical, is currently biosynthesized from D-xylose via a four-enzyme reaction cascade, with the ThDP-dependent α-keto acid decarboxylase (KdcA) identified as the potential bottleneck. Here, to further enhance the catalytic activity of KdcA toward the non-native substrate α-keto-3-deoxy-xylonate (KDX), in silico screening and structure-guided evolution were performed. The best mutants, S286L/G402P and V461K, exhibited a 1.8- and 2.5-fold higher enzymatic activity in the conversion of KDX to 3,4-dihydroxybutanal when compared to KdcA, respectively. MD simulations revealed that the two sets of mutations reshaped the substrate binding pocket, thereby increasing the binding affinity for KDX and promoting interactions between KDX and cofactor ThDP. Then, when the V461K mutant instead of wild type KdcA was integrated into the enzyme cascade, a 1.9-fold increase in BTO titer was observed. After optimization of the reaction conditions, the enzyme cocktail contained V461K converted 60 g/L D-xylose to 22.1 g/L BTO with a yield of 52.1 %. This work illustrated that protein engineering is a powerful tool for modifying the output of metabolic pathway.

Structure, function, and evolution of metallo-ß-lactamases from the B3 subgroup-emerging targets to combat antibiotic resistance.

ß-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 ß-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 ß-lactamases to inactivate this class of antibiotics via hydrolysis. Although nucleophilic serine-ß-lactamases have long been clinically important, most broad-spectrum ß-lactamases employ one or two metal ions (likely Zn2+) in catalysis. To date, potent and clinically useful inhibitors of these metallo-ß-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 to the B1 subgroup. Most characterized B3 MBLs have been discovered in environmental bacteria, but they are increasingly identified in clinical samples. B3-type MBLs display greater diversity in their active sites than other MBLs. Furthermore, at least one of the known B3-type MBLs is inhibited by the serine-ß-lactamase inhibitor clavulanic acid, an observation that may promote the design of derivatives active against a broader range of MBLs. In this Mini Review, recent advances in structure-function relationships of B3-type MBLs will be discussed, with a view to inspiring inhibitor development to combat the growing spread of ß-lactam resistance.

Inhibitors for metallo-ß-lactamases from the B1 and B3 subgroups provide an avenue to combat a major mechanism of antibiotic resistance.

Metallo-ß-lactamases (MBLs) are a group of Zn(II)-dependent enzymes that pose a major threat to global health. They are linked to an increasing number of multi-drug resistant bacterial pathogens, but no clinically useful inhibitor is yet available. Since ß-lactam antibiotics, which are inactivated by MBLs, constitute ∼65% of all antibiotics used to treat infections, the search for clinically relevant MBL inhibitors is urgent. Here, derivatives of a 2-amino-1-benzyl-4,5-diphenyl-1H-pyrrole-3-carbonitrile (1a) were synthesised and their inhibitory effects assessed against prominent representatives of the MBL family. Several compounds are potent inhibitors of each MBL tested, making them promising candidates for the development of broad-spectrum drug leads. In particular, compound 5f is highly potent across the MBL family, with Ki values in the low µM range. Furthermore, this compound also appears to display synergy in combination with antibiotics such as penicillin G, cefuroxime or meropenem. This molecule thus represents a promising starting point to develop new drugs to inhibit a major mechanism of antibiotic resistance.

Biomimetics for purple acid phosphatases: A historical perspective.

Biomimetics hold potential for varied applications in biotechnology and medicine but have also attracted particular interest as benchmarks for the functional study of their more complex biological counterparts, e.g. metalloenzymes. While many of the synthetic systems adequately mimic some structural and functional aspects of their biological counterparts the catalytic efficiencies displayed are mostly far inferior due to the smaller size and the associated lower complexity. Nonetheless they play an important role in bioinorganic chemistry. Numerous examples of biologically inspired and informed artificial catalysts have been reported, designed to mimic a plethora of chemical transformations, and relevant examples are highlighted in reviews and scientific reports. Herein, we discuss biomimetics of the metallohydrolase purple acid phosphatase (PAP), examples of which have been used to showcase synergistic research advances for both the biological and synthetic systems. In particular, we focus on the seminal contribution of our colleague Prof. Ademir Neves, and his group, pioneers in the design and optimization of suitable ligands that mimic the active site of PAP.

LAM-1 from Lysobacter antibioticus: A potent zinc-dependent activity that inactivates ß-lactam antibiotics.

Resistance to ß-lactam antibiotics, including the "last-resort" carbapenems, has emerged as a major threat to global health. A major resistance mechanism employed by pathogens involves the use of metallo-ß-lactamases (MBLs), zinc-dependent enzymes that inactivate most of the ß-lactam antibiotics used to treat infections. Variants of MBLs are frequently discovered in clinical environments. However, an increasing number of such enzymes have been identified in microorganisms that are less impacted by human activities. Here, an MBL from Lysobacter antibioticus, isolated from the rhizosphere, has been shown to be highly active toward numerous ß-lactam antibiotics. Its activity is higher than that of some of the most effective MBLs linked to hospital-acquired antibiotic resistance and thus poses an interesting system to investigate evolutionary pressures that drive the emergence of such biocatalysts.

Sequence- and structure-guided improvement of the catalytic performance of a GH11 family xylanase from Bacillus subtilis.

Xylanases produce xylooligosaccharides from xylan and have thus attracted increasing attention for their usefulness in industrial applications. Previously, we demonstrated that the GH11 xylanase XynLC9 from Bacillus subtilis formed xylobiose and xylotriose as the major products with negligible production of xylose when digesting corncob-extracted xylan. Here, we aimed to improve the catalytic performance of XynLC9 via protein engineering. Based on the sequence and structural comparisons of XynLC9 with the xylanases Xyn2 from Trichoderma reesei and Xyn11A from Thermobifida fusca, we identified the N-terminal residues 5-YWQN-8 in XynLC9 as engineering hotspots and subjected this sequence to site saturation and iterative mutagenesis. The mutants W6F/Q7H and N8Y possessed a 2.6- and 1.8-fold higher catalytic activity than XynLC9, respectively, and both mutants were also more thermostable. Kinetic measurements suggested that W6F/Q7H and N8Y had lower substrate affinity, but a higher turnover rate (kcat), which resulted in increased catalytic efficiency than WT XynLC9. Furthermore, the W6F/Q7H mutant displayed a 160% increase in the yield of xylooligosaccharides from corncob-extracted xylan. Molecular dynamics simulations revealed that the W6F/Q7H and N8Y mutations led to an enlarged volume and surface area of the active site cleft, which provided more space for substrate entry and product release and thus accelerated the catalytic activity of the enzyme. The molecular evolution approach adopted in this study provides the design of a library of sequences that captures functional diversity in a limited number of protein variants.

Kinetic and Structural Characterization of the First B3 Metallo-ß-Lactamase with an Active-Site Glutamic Acid.

The structural diversity in metallo-ß-lactamases (MBLs), especially in the vicinity of the active site, has been a major hurdle in the development of clinically effective inhibitors. Representatives from three variants of the B3 MBL subclass, containing either the canonical HHH/DHH active-site motif (present in the majority of MBLs in this subclass) or the QHH/DHH (B3-Q) or HRH/DQK (B3-RQK) variations, were reported previously. Here, we describe the structure and kinetic properties of the first example (SIE-1) of a fourth variant containing the EHH/DHH active-site motif (B3-E). SIE-1 was identified in the hexachlorocyclohexane-degrading bacterium Sphingobium indicum, and kinetic analyses demonstrate that although it is active against a wide range of antibiotics, its efficiency is lower than that of other B3 MBLs but has increased efficiency toward cephalosporins relative to other ß-lactam substrates. The overall fold of SIE-1 is characteristic of the MBLs; the notable variation is observed in the Zn1 site due to the replacement of the canonical His116 by a glutamate. The unusual preference of SIE-1 for cephalosporins and its occurrence in a widespread environmental organism suggest the scope for increased MBL-mediated ß-lactam resistance. Thus, it is relevant to include SIE-1 in MBL inhibitor design studies to widen the therapeutic scope of much needed antiresistance drugs.

Enhancing the catalytic activity of a GH5 processive endoglucanase from Bacillus subtilis BS-5 by site-directed mutagenesis.

Processive endoglucanases possess both endo- and exoglucanase activity, making them attractive discovery and engineering targets. Here, a processive endoglucanase EG5C-1 from Bacillus subtilis was employed as the starting point for enzyme engineering. Referring to the complex structure information of EG5C-1 and cellohexaose, the amino acid residues in the active site architecture were identified and subjected to alanine scanning mutagenesis. The residues were chosen for a saturation mutagenesis since their variants showed similar activities to EG5C-1. Variants D70Q and S235W showed increased activity towards the substrates CMC and Avicel, an increase was further enhanced in D70Q/S235W double mutant, which displayed a 2.1- and 1.7-fold improvement in the hydrolytic activity towards CMC and Avicel, respectively. In addition, kinetic measurements showed that double mutant had higher substrate affinity (Km) and a significantly higher catalytic efficiency (kcat/Km). The binding isotherms of wild-type EG5C-1 and double mutant D70Q/S235W suggested that the binding capability of EG5C-1 for the insoluble substrate was weaker than that of D70Q/S235W. Molecular dynamics simulations suggested that the collaborative substitutions of D70Q and S235W altered the hydrogen bonding network within the active site architecture and introduced new hydrogen bonds between the enzyme and cellohexaose, thus enhancing both substrate affinity and catalytic efficiency.

Structure and mechanism of potent bifunctional ß-lactam- and homoserine lactone-degrading enzymes from marine microorganisms.

Genes that confer antibiotic resistance can rapidly be disseminated from one microorganism to another by mobile genetic elements, thus transferring resistance to previously susceptible bacterial strains. The misuse of antibiotics in health care and agriculture has provided a powerful evolutionary pressure to accelerate the spread of resistance genes, including those encoding ß-lactamases. These are enzymes that are highly efficient in inactivating most of the commonly used ß-lactam antibiotics. However, genes that confer antibiotic resistance are not only associated with pathogenic microorganisms, but are also found in non-pathogenic (i.e. environmental) microorganisms. Two recent examples are metal-dependent ß-lactamases (MBLs) from the marine organisms Novosphingobium pentaromativorans and Simiduia agarivorans. Previous studies have demonstrated that their ß-lactamase activity is comparable to those of well-known MBLs from pathogenic sources (e.g. NDM-1, AIM-1) but that they also possess efficient lactonase activity, an activity associated with quorum sensing. Here, we probed the structure and mechanism of these two enzymes using crystallographic, spectroscopic and fast kinetics techniques. Despite highly conserved active sites both enzymes demonstrate significant variations in their reaction mechanisms, highlighting both the extraordinary ability of MBLs to adapt to changing environmental conditions and the rather promiscuous acceptance of diverse substrates by these enzymes.

Adaptation of a continuous, calorimetric kinetic assay to study the agmatinase-catalyzed hydrolytic reaction.

Ureohydrolases are members of the metallohydrolase family of enzymes. Here, a simple continuous assay for agmatinase (AGM) activity was established by following the degradation of agmatine to urea and putrescine using isothermal titration calorimetry (ITC). ITC is particularly useful for kinetic assays when substrates of interest do not possess suitable chromophores that facilitate the continuous spectrophotometric detection of substrate depletion and/or product formation. In order to assess the accuracy of the ITC-based assay, catalytic parameters were also determined using a discontinuous, colorimetric assay. Both methods resulted in comparable kinetic parameters. From the colorimetric assay the kcat and KM values are 131 s-1 and 0.25 mM, respectively, and from the ITC assay the corresponding parameters are 30 s-1 and 0.45 mM, respectively. The continuous ITC-based assay will facilitate functional studies for an enzyme that is an emerging target for the development of addiction treatments.

Expansin assisted bio-affinity immobilization of endoxylanase from Bacillus subtilis onto corncob residue: Characterization and efficient production of xylooligosaccharides.

A one-step method to immobilize xylanase onto cellulosic material by fusion of expansin from Bacillus subtilis to xylanase LC9 without the requirement of prior purification of enzyme has been developed. Fusion enzyme EXLX-R2-XYN was specifically adsorbed onto corncob residue with high loading capacity due to bio-affinity adsorption of expansin onto cellulose. The immobilization yield was close to 100%, with a recovered activity of 82.4%. The immobilized EXLX-R2-XYN retained 45.3% of its activity after incubation at 70 °C for 3 h, whereas only 16.3% of the activity was left in free form under the same conditions. The conversion yield of XOS by using immobilized EXLX-R2-XYN reached up to 515 mg/g xylan from 2% corncob extracted xylan, which was higher than that of the free enzyme. The hydrolysis products were mainly xylobiose (57.5%) and xylotriose (38.4%), without undesirable xylose production. After five cycles of hydrolysis, more than 70% of conversion was obtained.

Relative catalytic efficiencies and transcript levels of three d- and two l-lactate dehydrogenases for optically pure d-lactate production in Sporolactobacillus inulinus.

As the optical purity of the lactate monomer is pivotal for polymerization, the production of optically pure d-lactate is of significant importance. Sporolactobacillus inulinus YBS1-5 is a superior optically pure d-lactate-producing bacterium. However, little is known about the relationship between lactate dehydrogenases in S. inulinus YBS1-5 and the optical purity of d-lactate. Three potential d-lactate dehydrogenase (D-LDH1-3)- and two putative l-lactate dehydrogenase (L-LDH1-2)-encoding genes were cloned from the YBS1-5 strain and expressed in Escherichia coli D-LDH1 exhibited the highest catalytic efficiency toward pyruvate, whereas two L-LDHs showed low catalytic efficiency. Different neutralizers significantly affected the optical purity of d-lactate produced by strain YBS1-5 as well as the transcription levels of ldhDs and ldhLs. The high catalytic efficiency of D-LDH1 and elevated ldhD1 mRNA levels suggest that this enzyme is essential for d-lactate synthesis in S. inulinus YBS1-5. The correlation between the optical purity of d-lactate and transcription levels of ldhL1 in the case of different neutralizers indicate that ldhL1 is a key factor affecting the optical purity of d-lactate in S. inulinus YBS1-5.

Phytochemical and antimicrobial study of Pilocarpus pennatifolius Lemaire.

The investigation of the crude extract of leaves and bark of Pilocarpus pennatifolius Lemaire allowed isolated of a not yet described coumarin, together with three known coumarins (bergapten, xanthotoxin and dimethyl allyl xanthyletin), and a not yet described imidazole alkaloid. All structures were established by means of spectral analysis, including extensive 2D NMR studies. In addition, the alkaloid had its absolute stereochemistry determined by X-ray diffraction. Meanwhile, extracts and pure compounds were tested against various strains of bacteria and fungi, showing promising antimicrobial activities. We highlight the activities of crude bark methanol extract (CBME), of the leaf basic acetate fraction (LBAcF), and of compound 2 against the Gram negative bacteria Shigella flexneri (MICs = 7.8, 7.8 and 3.12 µg·mL-1, respectively), of compound 5 against the Gram positive Enterococcus fecalis (MIC = 1.56 µg·mL-1), and against two Gram negative bacteria Salmonella enteritidis (MIC = 1.56 µg·mL-1), and Pseudomonas aeruginosa (MIC = 6.25 µg·ml-1). On the other hand, CBME and compounds 3-5 showed excellent activity against the fungus Candida krusei with MICs of 15.6, 1.56, and 3.12 µg·mL-1 respectively, as actives or better than the antifungal standard fluconazole (MIC = 3.12 µg·mL-1).

Processivity and enzymatic mechanism of a multifunctional family 5 endoglucanase from Bacillus subtilis BS-5 with potential applications in the saccharification of cellulosic substrates.

BACKGROUND: Presently, 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. RESULTS: The 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-ß-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. CONCLUSIONS: The 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.

Characterization of a highly efficient antibiotic-degrading metallo-ß-lactamase obtained from an uncultured member of a permafrost community.

Antibiotic resistance is a major global health problem, one that threatens to derail the benefits garnered from arguably the greatest success of modern medicine, the discovery of antibiotics. Among the most potent agents contributing to antibiotic resistance are metallo-ß-lactamases (MBLs). The discovery of MBL-like enzymes in microorganisms that are not in contact with the human population is of particular concern as these proteins already have the in-built capacity to inactivate antibiotics, even though they may not need MBL activity for their survival. Here, we demonstrate that a microbiome from a remote and frozen environment in Alaska harbours at least one highly efficient MBL, LRA-8. LRA-8 is homologous to the B3 subgroup of MBLs and has a substrate profile and catalytic properties similar to well-known members of this enzyme family, which are expressed by major human pathogens. LRA-8 is predominantly a penicillinase, but is also active towards carbapenems, but not cephalosporins. Spectroscopic studies indicate that LRA-8 has an active site structure similar to that of other MBLs (in particular B3 subgroup representative AIM-1), and a combination of steady-state and pre-steady-state kinetic data demonstrate that the enzyme is likely to employ a metal ion-bridging hydroxide to initiate catalysis. The rate-limiting step is the decay of a chromophoric, tetrahedral intermediate, as is observed in various other MBLs. Thus, studying the properties of such "pristine" MBL-like proteins may provide insight into the structural plasticity of this family of enzymes that may facilitate functional promiscuity, while important insight into the evolution of MBLs may also be gained.

High resolution crystal structure of a fluoride-inhibited organophosphate-degrading metallohydrolase.

Metal ion-dependent, organophosphate-degrading enzymes (OP hydrolases) have received increasing attention due to their ability to degrade and thus detoxify commonly used pesticides and nerve agents such as sarin and VX. These enzymes thus garner strong potential as bioremediators. The OP hydrolase from Agrobacterium radiobacter (OpdA) is one of the most efficient members of this group of enzymes. Previous studies have indicated that the choice of the hydrolysis-initiating nucleophile may depend on the pH of the reaction, with a metal ion-bridging hydroxide being preferred at lower pH (i.e. pH≤8.5), and a terminally coordinated hydroxide at higher pH (i.e. pH>9.0). Furthermore, fluoride was shown to be a potent inhibitor of the reaction, but only at low pH. Here, the crystal structure (1.3Å, pH6) of OpdA in presence of fluoride is described. While the first coordination sphere in the active site displays minimal changes in the presence of fluoride, the hydrogen bonding network that connects the dimetallic metal center to the substrate binding pocket is disrupted. Thus, the structure of fluoride-inhibited OpdA demonstrates the significance of this hydrogen bond network in controlling the mechanism and function of this enzyme.

Phytochemical analysis of bark from Helietta apiculata Benth and antimicrobial activities.

Extraction and characterization of natural products from the bark of the trunk of Helietta apiculata Benth (Rutaceae) afforded nine alkaloids, eight furoquinoline and one quinolone, limonine, three cinnamic acid derivatives, three neolignans, tetracosanoic acid, six coumarins, of which apiculin A and apiculin B (neolignans), and tanizin (coumarin) are previously undescribed compounds. The structures of all compounds were determined by spectroscopic methods, and the crystal structures of two of the newly undescribed compounds, apiculin A and apiculin B, were determined by X-ray analysis. Extracts and pure compounds isolated from Helietta apiculata showed promising antimicrobial activities.

Structure-activity relationship study and optimisation of 2-aminopyrrole-1-benzyl-4,5-diphenyl-1H-pyrrole-3-carbonitrile as a broad spectrum metallo-ß-lactamase inhibitor.

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-ß-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.