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.

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

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.

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

Metallo-β-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 β-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.

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

Resistance to β-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-β-lactamases (MBLs), zinc-dependent enzymes that inactivate most of the β-lactam antibiotics used to treat infections. Variants of MBLs are frequently discovered in clinical environments.

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.

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

The structural diversity in metallo-β-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).

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.

Structure and mechanism of potent bifunctional β-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 β-lactamases. These are enzymes that are highly efficient in inactivating most of the commonly used β-lactam antibiotics.

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.

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.

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.

Processivity and enzymatic mechanism of a multifunctional family 5 endoglucanase from 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.

Characterization of a highly efficient antibiotic-degrading metallo-β-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-β-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.

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.

Structure-activity relationship study and optimisation of 2-aminopyrrole-1-benzyl-4,5-diphenyl-1H-pyrrole-3-carbonitrile as a broad spectrum metallo-β-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-β-lactamases (MBLs), i.e. IMP-1 (representing the B1 subgroup), CphA (B2) and AIM-1 (B3).

Reaction mechanism of the metallohydrolase CpsB from Streptococcus pneumoniae, a promising target for novel antimicrobial agents.

CpsB is a metal ion-dependent hydrolase involved in the biosynthesis of capsular polysaccharides in bacterial organisms. The enzyme has been proposed as a promising target for novel chemotherapeutics to combat antibiotic resistance. The crystal structure of CpsB indicated the presence of as many as three closely spaced metal ions, modelled as Mn, in the active site.

Visualization of the Reaction Trajectory and Transition State in a Hydrolytic Reaction Catalyzed by a Metalloenzyme.

Metallohydrolases are a vast family of enzymes that play crucial roles in numerous metabolic pathways. Several members have emerged as targets for chemotherapeutics. Knowledge about their reaction mechanisms and associated transition states greatly aids the design of potent and highly specific drug leads.

Insights into an evolutionary strategy leading to antibiotic resistance.

Metallo-β-lactamases (MBLs) with activity towards a broad-spectrum of β-lactam antibiotics have become a major threat to public health, not least due to their ability to rapidly adapt their substrate preference. In this study, the capability of the MBL AIM-1 to evade antibiotic pressure by introducing specific mutations was probed by two alternative methods, i.e.

AIM-1: An Antibiotic-Degrading Metallohydrolase That Displays Mechanistic Flexibility.

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.