Protein metal-coordination sites are richly varied and exquisitely attuned to their inorganic partners, yet many metalloproteins still select the wrong metals when presented with mixtures of ...elements. Cells have evolved elaborate mechanisms to scavenge for sufficient metal atoms to meet their needs and to adjust their needs to match supply. Metal sensors, transporters and stores have often been discovered as metal-resistance determinants, but it is emerging that they perform a broader role in microbial physiology: they allow cells to overcome inadequate protein metal affinities to populate large numbers of metalloproteins with the right metals.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Haemophilia A severity is closely correlated to the factor VIII (FVIII) activity, which can be measured in different ways. The original one‐stage clotting assay is still the most widely used. The ...two‐stage coagulation assay eliminated many of the drawbacks of the one‐stage assay and was further developed into the chromogenic assay, a two‐staged test with purified coagulation factors in the first stage, and a FXa‐specific chromogenic substrate in the second stage. In many patients with mild or moderate haemophilia A, there is a discrepancy between the one‐stage and the two‐stage assays. If only the one‐stage assay is used, some patients will have normal FVIII levels and not be diagnosed as having haemophilia or be considered to have a milder bleeding risk than is the case. Other patients who have normal FVIII activity will be diagnosed as haemophilia A. All haemophilia treatment centre laboratories should have access to both one‐stage and chromogenic FVIII:C assays. Appropriate standards should be employed to enable accurate FVIII:C measurement. Different assays to measure inhibitor activity to infused FVIII have been developed since 1959. Inhibitor results based on the one‐stage or chromogenic FVIII:C assays are well correlated, but the one‐stage assay may be influenced by nonspecific inhibition.
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BFBNIB, DOBA, FZAB, GIS, IJS, IZUM, KILJ, NLZOH, NUK, OILJ, PILJ, PNG, SAZU, SBCE, SBMB, SIK, UILJ, UKNU, UL, UM, UPUK
Blomberg et al present information regarding quantum chemical studies of mechanisms for metalloenzymes, focusing on heme enzymes, non-heme iron enzymes, and copper enzymes. Other topics addressed ...include hydrogenase, nitrogenase, and methyl-coenzynme M reductase.
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Enzymes are ideal for use in asymmetric catalysis by the chemical industry, because their chemical compositions can be tailored to a specific substrate and selectivity pattern while providing ...efficiencies and selectivities that surpass those of classical synthetic methods. However, enzymes are limited to reactions that are found in nature and, as such, facilitate fewer types of transformation than do other forms of catalysis. Thus, a longstanding challenge in the field of biologically mediated catalysis has been to develop enzymes with new catalytic functions. Here we describe a method for achieving catalytic promiscuity that uses the photoexcited state of nicotinamide co-factors (molecules that assist enzyme-mediated catalysis). Under irradiation with visible light, the nicotinamide-dependent enzyme known as ketoreductase can be transformed from a carbonyl reductase into an initiator of radical species and a chiral source of hydrogen atoms. We demonstrate this new reactivity through a highly enantioselective radical dehalogenation of lactones-a challenging transformation for small-molecule catalysts. Mechanistic experiments support the theory that a radical species acts as an intermediate in this reaction, with NADH and NADPH (the reduced forms of nicotinamide adenine nucleotide and nicotinamide adenine dinucleotide phosphate, respectively) serving as both a photoreductant and the source of hydrogen atoms. To our knowledge, this method represents the first example of photo-induced enzyme promiscuity, and highlights the potential for accessing new reactivity from existing enzymes simply by using the excited states of common biological co-factors. This represents a departure from existing light-driven biocatalytic techniques, which are typically explored in the context of co-factor regeneration.
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Multiple mitochondrial matrix enzymes playing key roles in metabolism require cofactors for their action. Due to the high impermeability of the mitochondrial inner membrane, these cofactors need to ...be synthesized within the mitochondria or be imported, themselves or one of their precursors, into the organelles. Transporters belonging to the protein family of mitochondrial carriers have been identified to transport the coenzymes: thiamine pyrophosphate, coenzyme A, FAD and NAD+, which are all structurally similar to nucleotides and derived from different B‐vitamins. These mitochondrial cofactors bind more or less tightly to their enzymes and, after having been involved in a specific reaction step, are regenerated, spontaneously or by other enzymes, to return to their active form, ready for the next catalysis round. Disease‐causing mutations in the mitochondrial cofactor carrier genes compromise not only the transport reaction but also the activity of all mitochondrial enzymes using that particular cofactor and the metabolic pathways in which the cofactor‐dependent enzymes are involved. The mitochondrial transport, metabolism and diseases of the cofactors thiamine pyrophosphate, coenzyme A, FAD and NAD+ are the focus of this review.
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FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SBCE, SBMB, UL, UM, UPUK
Protein lysine methylation is a distinct posttranslational modification that causes minimal changes in the size and electrostatic status of lysine residues. Lysine methylation plays essential roles ...in regulating fates and functions of target proteins in an epigenetic manner. As a result, substrates and degrees (free versus mono/di/tri) of protein lysine methylation are orchestrated within cells by balanced activities of protein lysine methyltransferases (PKMTs) and demethylases (KDMs). Their dysregulation is often associated with neurological disorders, developmental abnormalities, or cancer. Methyllysine-containing proteins can be recognized by downstream effector proteins, which contain methyllysine reader domains, to relay their biological functions. While numerous efforts have been made to annotate biological roles of protein lysine methylation, limited work has been done to uncover mechanisms associated with this modification at a molecular or atomic level. Given distinct biophysical and biochemical properties of methyllysine, this review will focus on chemical and biochemical aspects in addition, recognition, and removal of this posttranslational mark. Chemical and biophysical methods to profile PKMT substrates will be discussed along with classification of PKMT inhibitors for accurate perturbation of methyltransferase activities. Semisynthesis of methyllysine-containing proteins will also be covered given the critical need for these reagents to unambiguously define functional roles of protein lysine methylation.
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The structure of hexameric glutamate dehydrogenase (GDH) in the presence of the coenzyme nicotinamide adenine dinucleotide phosphate (NADP) was visualized using cryogenic transmission electron ...microscopy to investigate the ligand‐binding pathways to the active site of the enzyme. Each subunit of GDH comprises one hexamer‐forming core domain and one nucleotide‐binding domain (NAD domain), which spontaneously opens and closes the active‐site cleft situated between the two domains. In the presence of NADP, the potential map of GDH hexamer, assuming D3 symmetry, was determined at a resolution of 2.4 Å, but the NAD domain was blurred due to the conformational variety. After focused classification with respect to the NAD domain, the potential maps interpreted as NADP molecules appeared at five different sites in the active‐site cleft. The subunits associated with NADP molecules were close to one of the four metastable conformations in the unliganded state. Three of the five binding sites suggested a pathway of NADP molecules to approach the active‐site cleft for initiating the enzymatic reaction. The other two binding modes may rarely appear in the presence of glutamate, as demonstrated by the reaction kinetics. Based on the visualized structures and the results from the enzymatic kinetics, we discussed the binding modes of NADP to GDH in the absence and presence of glutamate.
Binding pathways of nicotinamide adenine dinucleotide phosphate (NADP) to the active‐site cleft of glutamate dehydrogenase (GDH) were studied by cryo‐EM for GDH‐NADP mixture. Three of the five binding sites suggested a pathway of NADP to approach the position suitable for enzymatic reaction in the active‐site cleft. NADP molecules in the other two sites were estimated as nonproductive and rare in the presence of glutamate.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SBCE, SBMB, UL, UM, UPUK
The title compound, a key intermediate for preparing Coenzyme Qn family, was prepared in high yield by a reaction sequence starting from the commercially available 3, 4, 5-trimethoxy-benzadehyde via ...Wolff-Kishner reduction, Vilsmeier-Haack reaction, Blanc chloromethylation reaction, Dakin reaction and oxidation.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
Enzymatic immobilization has been at the forefront of applied biocatalysis as it enables convenient isolation and reuse of the catalyst if the target reaction is conducted in batch, and it has opened ...up significant opportunities to conduct biocatalysis in continuous mode. Over the last few years, an array of techniques to immobilize enzymes have been developed, spanning from covalent multipoint attachment to noncovalent electrostatic strategies to rational architecture to suitably orient the enzyme(s). In addition, new materials have been adapted to support biological catalysts. Here, we discuss the advances of the last two years in enzyme immobilization for continuous flow applications.
•Enzyme encapsulation in solgel matrices, 3D-printing, and all-enzyme hydrogels.•Metal affinity immobilization of polyhistidine tag enzymes on EziG™ and vortex fluidic devices.•Covalent immobilization of multienzymatic cascade systems in tandem reactors.•Coimmobilization of multienzymatic systems and redox cofactors.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
DNAmoreDB, a database of DNAzymes Ponce-Salvatierra, Almudena; Boccaletto, Pietro; Bujnicki, Janusz M
Nucleic acids research,
01/2021, Volume:
49, Issue:
D1
Journal Article
Peer reviewed
Open access
Abstract
Deoxyribozymes, DNA enzymes or simply DNAzymes are single-stranded oligo-deoxyribonucleotide molecules that, like proteins and ribozymes, possess the ability to perform catalysis. Although ...DNAzymes have not yet been found in living organisms, they have been isolated in the laboratory through in vitro selection. The selected DNAzyme sequences have the ability to catalyze a broad range of chemical reactions, utilizing DNA, RNA, peptides or small organic compounds as substrates. DNAmoreDB is a comprehensive database resource for DNAzymes that collects and organizes the following types of information: sequences, conditions of the selection procedure, catalyzed reactions, kinetic parameters, substrates, cofactors, structural information whenever available, and literature references. Currently, DNAmoreDB contains information about DNAzymes that catalyze 20 different reactions. We included a submission form for new data, a REST-based API system that allows users to retrieve the database contents in a machine-readable format, and keyword and BLASTN search features. The database is publicly available at https://www.genesilico.pl/DNAmoreDB/.
Graphical Abstract
Graphical Abstract
DNAmoreDB, a comprehensive database resource for DNAzymes.
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