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.

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.

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.

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.

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.

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.

Metal Ions Play an Essential Catalytic Role in the Mechanism of Ketol-Acid Reductoisomerase.

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.

Ca(II) Binding Regulates and Dominates the Reactivity of a Transition-Metal-Ion-Dependent Diesterase from Mycobacterium tuberculosis.

The diesterase Rv0805 from Mycobacterium tuberculosis is a dinuclear metallohydrolase that plays an important role in signal transduction by controlling the intracellular levels of cyclic nucleotides. As Rv0805 is essential for mycobacterial growth it is a promising new target for the development of chemotherapeutics to treat tuberculosis. The in vivo metal-ion composition of Rv0805 is subject to debate.

Catalytic mechanisms of metallohydrolases containing two metal ions.

At least one-third of enzymes contain metal ions as cofactors necessary for a diverse range of catalytic activities. In the case of polymetallic enzymes (i.e.

Comparative investigation of the reaction mechanisms of the organophosphate-degrading phosphotriesterases from Agrobacterium radiobacter (OpdA) and Pseudomonas diminuta (OPH).

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.

Structural and functional analysis of a FeoB A143S G5 loop mutant explains the accelerated GDP release rate.

GTPases (G proteins) hydrolyze the conversion of GTP to GDP and free phosphate, comprising an integral part of prokaryotic and eukaryotic signaling, protein biosynthesis and cell division, as well as membrane transport processes. The G protein cycle is brought to a halt after GTP hydrolysis, and requires the release of GDP before a new cycle can be initiated. For eukaryotic heterotrimeric Gαβγ proteins, the interaction with a membrane-bound G protein-coupled receptor catalyzes the release of GDP from the Gα subunit.

Determination of the catalytic activity of binuclear metallohydrolases using isothermal titration calorimetry.

Binuclear metallohydrolases are a large and diverse family of enzymes that are involved in numerous metabolic functions. An increasing number of members find applications as drug targets or in processes such as bioremediation. It is thus essential to have an assay available that allows the rapid and reliable determination of relevant catalytic parameters (k cat, K m, and k cat/K m).

Phosphate-bound structure of an organophosphate-degrading enzyme from Agrobacterium radiobacter.

OpdA is a binuclear metalloenzyme that can hydrolyze organophosphate pesticides and nerve agents. In this study the crystal structure of the complex between OpdA and phosphate has been determined to 2.20 Å resolution.