scholarly journals Glyoxylate reductase as versatile enzymatic system for pharmaceutical and medical use

2016 ◽  
Author(s):  
Jan Kutner ◽  
Ivan Shabalin ◽  
Dorota Matelska ◽  
Katarzyna B Handing ◽  
Olga Gasiorowska ◽  
...  
Keyword(s):  
Pharmaceutical Industry ◽  
Substrate Specificity ◽  
Large Scale ◽  
Catalytic Domain ◽  
Enzyme Stability ◽  
Industrial Process ◽  
Binding Domain ◽  
Enzymatic System ◽  
Dependent Manner ◽  
Glyoxylate Reductase

The chiral homogeneity of a chemical compound is the main prerequisite in safety and efficiency of drug substances and generation of single enantiomers of drug intermediates in pharmaceutical industry. Over the past several years there have been an increase wide variety of enzymes and bioengineered microorganisms used for biotransformation of chemicals with chemo- regio- and enantioselectivity. The direct evolution – a combination of biochemistry, molecular biology, structural biology and bioinformatics predictions can modulate enzyme stability, reactivity or substrate specificity. One example of enzymes used in pharmaceutical industry are enzymes from the D-2-hydroxyacid dehydrogenase (2HADH) family. They catalyze reversible reduction of 2-oxoacids to 2-hydroxyacids in an NAD(P)H dependent manner, playing a key role in metabolism of many organisms. One of the enzyme group from 2HADH family is glyoxylate reductase (GR). The advantages of these enzymes have been recently recognized by pharmaceutical and biotechnological industry, considering a possibility of highly stereospecific biotransformation of α-ketoacids into homochiral α-hydroxyacids, as important industrial intermediates. The glycolic acid, reduced by the enzyme into ethylene glycol, the smallest member of the α-hydroxy acid family, is nowadays obtained in a large-scale industrial process. The monomeric structure of the enzyme comprises two domains typical for NAD(P)-dependent dehydrogenases: the substrate-binding domain (SBD) and the nucleotide-binding domain (NBD). Several crystal X-ray structures of glyoxylate reductase from different species have been already solved, but only few of them were determined with the bound substrate and/or cofactor. Such structures are crucial in understanding the reaction mechanism and for predicting or designing the structures of new substrates for the enzyme. We will communicate the structural study of two homodimeric glyoxylate reductases from S. meliloti (SmGR1 and SmGR2). We have solved the crystal structures for SmGR1 and SmGR2 with bound oxalate and NADPH and compared them with the structures of other glyoxylate reductases subfamily members: from Rhizobium etli (with L(+)-tartaric acid and NADP) and human (with (2 R )-2,3-dihydroxypropanoic acid and NADPH). During the ligand and cofactor binding the catalytic domain rotates towards coenzyme-binding site, changing its structure from open to close conformation. Using biochemical tools and kinetics approaches we have also shown that Sm GR1 and Sm GR2 possess substrate specificity to hydroxypuryvate, hydroxylphenylpyruvate and gloxylate, providing new insights into the potential pharmaceutical and medical uses of this family of enzymes.

scholarly journals Glyoxylate reductase as versatile enzymatic system for pharmaceutical and medical use

2016 ◽  
Author(s):  
Jan Kutner ◽  
Ivan Shabalin ◽  
Dorota Matelska ◽  
Katarzyna B Handing ◽  
Olga Gasiorowska ◽  
...  
Keyword(s):  
Pharmaceutical Industry ◽  
Substrate Specificity ◽  
Large Scale ◽  
Catalytic Domain ◽  
Enzyme Stability ◽  
Industrial Process ◽  
Binding Domain ◽  
Enzymatic System ◽  
Dependent Manner ◽  
Glyoxylate Reductase

The chiral homogeneity of a chemical compound is the main prerequisite in safety and efficiency of drug substances and generation of single enantiomers of drug intermediates in pharmaceutical industry. Over the past several years there have been an increase wide variety of enzymes and bioengineered microorganisms used for biotransformation of chemicals with chemo- regio- and enantioselectivity. The direct evolution – a combination of biochemistry, molecular biology, structural biology and bioinformatics predictions can modulate enzyme stability, reactivity or substrate specificity. One example of enzymes used in pharmaceutical industry are enzymes from the D-2-hydroxyacid dehydrogenase (2HADH) family. They catalyze reversible reduction of 2-oxoacids to 2-hydroxyacids in an NAD(P)H dependent manner, playing a key role in metabolism of many organisms. One of the enzyme group from 2HADH family is glyoxylate reductase (GR). The advantages of these enzymes have been recently recognized by pharmaceutical and biotechnological industry, considering a possibility of highly stereospecific biotransformation of α-ketoacids into homochiral α-hydroxyacids, as important industrial intermediates. The glycolic acid, reduced by the enzyme into ethylene glycol, the smallest member of the α-hydroxy acid family, is nowadays obtained in a large-scale industrial process. The monomeric structure of the enzyme comprises two domains typical for NAD(P)-dependent dehydrogenases: the substrate-binding domain (SBD) and the nucleotide-binding domain (NBD). Several crystal X-ray structures of glyoxylate reductase from different species have been already solved, but only few of them were determined with the bound substrate and/or cofactor. Such structures are crucial in understanding the reaction mechanism and for predicting or designing the structures of new substrates for the enzyme. We will communicate the structural study of two homodimeric glyoxylate reductases from S. meliloti (SmGR1 and SmGR2). We have solved the crystal structures for SmGR1 and SmGR2 with bound oxalate and NADPH and compared them with the structures of other glyoxylate reductases subfamily members: from Rhizobium etli (with L(+)-tartaric acid and NADP) and human (with (2 R )-2,3-dihydroxypropanoic acid and NADPH). During the ligand and cofactor binding the catalytic domain rotates towards coenzyme-binding site, changing its structure from open to close conformation. Using biochemical tools and kinetics approaches we have also shown that Sm GR1 and Sm GR2 possess substrate specificity to hydroxypuryvate, hydroxylphenylpyruvate and gloxylate, providing new insights into the potential pharmaceutical and medical uses of this family of enzymes.

The Effect of Active Site-inhibited Factor VIIa on Tissue Factor-initiated Coagulation Using Platelets before and after Aspirin Administration

1997 ◽  
Vol 78 (04) ◽  
pp. 1202-1208 ◽  
Author(s):  
Marianne Kjalke ◽  
Julie A Oliver ◽  
Dougald M Monroe ◽  
Maureane Hoffman ◽  
Mirella Ezban ◽  
...  
Keyword(s):  
Tissue Factor ◽  
Active Site ◽  
Large Scale ◽  
Thrombin Generation ◽  
Factor Viia ◽  
Dependent Manner ◽  
Antithrombotic Agent ◽  
Before And After ◽  
Ic50 Values

SummaryActive site-inactivated factor VIIa has potential as an antithrombotic agent. The effects of D-Phe-L-Phe-L-Arg-chloromethyl ketone-treated factor VIla (FFR-FVIIa) were evaluated in a cell-based system mimicking in vivo initiation of coagulation. FFR-FVIIa inhibited platelet activation (as measured by expression of P-selectin) and subsequent large-scale thrombin generation in a dose-dependent manner with IC50 values of 1.4 ± 0.8 nM (n = 8) and 0.9 ± 0.7 nM (n = 7), respectively. Kd for factor VIIa binding to monocytes ki for FFR-FVIIa competing with factor VIIa were similar (11.4 ± 0.8 pM and 10.6 ± 1.1 pM, respectively), showing that FFR-FVIIa binds to tissue factor in the tenase complex with the same affinity as factor VIIa. Using platelets from volunteers before and after ingestion of aspirin (1.3 g), there were no significant differences in the IC50 values of FFR-FVIIa [after aspirin ingestion, the IC50 values were 1.7 ± 0.9 nM (n = 8) for P-selectin expression, p = 0.37, and 1.4 ± 1.3 nM (n = 7) for thrombin generation, p = 0.38]. This shows that aspirin treatment of platelets does not influence the inhibition of tissue factor-initiated coagulation by FFR-FVIIa, probably because thrombin activation of platelets is not entirely dependent upon expression of thromboxane A2.

Characterization of a novel moderate-substrate specificity amino acid racemase from the hyperthermophilic archaeon Thermococcus litoralis

2021 ◽  
Author(s):  
Ryushi Kawakami ◽  
Chinatsu Kinoshita ◽  
Tomoki Kawase ◽  
Mikio Sato ◽  
Junji Hayashi ◽  
...  
Keyword(s):  
Amino Acid ◽  
Substrate Specificity ◽  
Enzyme Stability ◽  
Hyperthermophilic Archaea ◽  
Thermococcus Litoralis ◽  
Hyperthermophilic Archaeon ◽  
Aminobutyrate Aminotransferase ◽  
Chaperone Protein ◽  
Amino Acid Racemase

Abstract The amino acid sequence of the OCC_10945 gene product from the hyperthermophilic archaeon Thermococcus litoralis DSM5473, originally annotated as γ-aminobutyrate aminotransferase, is highly similar to that of the uncharacterized pyridoxal 5ʹ-phosphate (PLP)-dependent amino acid racemase from Pyrococcus horikoshii. The OCC_10945 enzyme was successfully overexpressed in Escherichia coli by co-expression with a chaperone protein. The purified enzyme demonstrated PLP-dependent amino acid racemase activity primarily toward Met and Leu. Although PLP contributed to enzyme stability, it only loosely bound to this enzyme. Enzyme activity was strongly inhibited by several metal ions, including Co2+ and Zn2+, and non-substrate amino acids such as l-Arg and l-Lys. These results suggest that the underlying PLP-binding and substrate recognition mechanisms in this enzyme are significantly different from those of the other archaeal and bacterial amino acid racemases. This is the first description of a novel PLP-dependent amino acid racemase with moderate substrate specificity in hyperthermophilic archaea.

scholarly journals Potentiating Effect of Mandelate and Lactate on Chemically Induced Germination in Members of Bacillus cereus Sensu Lato

2017 ◽  
Vol 83 (24) ◽  
Author(s):  
Alistair H. Bishop
Keyword(s):  
Bacillus Cereus ◽  
Large Scale ◽  
Germination Rate ◽  
Dependent Manner ◽  
Clostridium Sporogenes ◽  
Specific Chemical ◽  
Content Type ◽  
Ph Range ◽  
Significant Discovery ◽  
The Impact

ABSTRACT Endospores of the genus Bacillus can be triggered to germinate by a limited number of chemicals. Mandelate had powerful additive effects on the levels and rates of germination produced in non-heat-shocked spores of Bacillus anthracis strain Sterne, Bacillus cereus, and Bacillus thuringiensis when combined with l-alanine and inosine. Mandelate had no germinant effect on its own but was active with these germinants in a dose-dependent manner at concentrations higher than 0.5 mM. The maximum rate and extent of germination were produced in B. anthracis by 100 mM l-alanine with 10 mM inosine; this was equaled by just 25% of these germinants when supplemented with 10 mM mandelate. Half the maximal germination rate was produced by 40% of the optimum germinant concentrations or 15% of them when supplemented with 0.8 mM mandelate. Germination rates in B. thuringiensis were highest around neutrality, but the potentiating effect of mandelate was maintained over a wider pH range than was germination with l-alanine and inosine alone. For all species, lactate also promoted germination in the presence of l-alanine and inosine; this was further increased by mandelate. Ammonium ions also enhanced l-alanine- and inosine-induced germination but only when mandelate was present. In spite of the structural similarities, mandelate did not compete with phenylalanine as a germinant. Mandelate appeared to bind to spores while enhancing germination. There was no effect when mandelate was used in conjunction with nonnutrient germinants. No effect was produced with spores of Bacillus subtilis, Clostridium sporogenes, or C. difficile. IMPORTANCE The number of chemicals that can induce germination in the species related to Bacillus cereus has been defined for many years, and they conform to specific chemical types. Although not a germinant itself, mandelate has a structure that is different from these germination-active compounds, and its addition to this list represents a significant discovery in the fundamental biology of spore germination. This novel activity may also have important applied relevance given the impact of spores of B. cereus in foodborne disease and B. anthracis as a threat agent. The destruction of spores of B. anthracis, for example, particularly over large outdoor areas, poses significant scientific and logistical problems. The addition of mandelate and lactate to the established mixtures of l-alanine and inosine would decrease the amount of the established germinants required and increase the speed and level of germination achieved. The large-scale application of “germinate to decontaminate” strategy may thus become more practicable.

scholarly journals Acetylene hydrochlorination using Au/carbon: a journey towards single site catalysis

2017 ◽  
Vol 53 (86) ◽  
pp. 11733-11746 ◽  
Author(s):  
Grazia Malta ◽  
Simon J. Freakley ◽  
Simon A. Kondrat ◽  
Graham J. Hutchings
Keyword(s):  
Environmental Impact ◽  
Vinyl Chloride ◽  
Mercuric Chloride ◽  
Large Scale ◽  
Industrial Process ◽  
Single Site ◽  
Vinyl Chloride Monomer ◽  
Acetylene Hydrochlorination

The replacement of mercuric chloride in the production of vinyl chloride monomer, a precursor to PVC, would greatly reduce the environmental impact of this large scale industrial process.

scholarly journals The Amino Terminus of Varicella-Zoster Virus (VZV) Glycoprotein E Is Required for Binding to Insulin-Degrading Enzyme, a VZV Receptor

2007 ◽  
Vol 81 (16) ◽  
pp. 8525-8532 ◽  
Author(s):  
Qingxue Li ◽  
Tammy Krogmann ◽  
Mir A. Ali ◽  
Wei-Jen Tang ◽  
Jeffrey I. Cohen
Keyword(s):  
Amino Acids ◽  
Varicella Zoster Virus ◽  
Catalytic Domain ◽  
Amino Terminus ◽  
Dependent Manner ◽  
Insulin Degrading Enzyme ◽  
Concentration Dependent Manner ◽  
Glycoprotein E ◽  
Degrading Enzyme ◽  
Varicella Zoster

ABSTRACT Varicella-zoster virus (VZV) glycoprotein E (gE) is required for VZV infection. Although gE is well conserved among alphaherpesviruses, the amino terminus of VZV gE is unique. Previously, we showed that gE interacts with insulin-degrading enzyme (IDE) and facilitates VZV infection and cell-to-cell spread of the virus. Here we define the region of VZV gE required to bind IDE. Deletion of amino acids 32 to 71 of gE, located immediately after the predicted signal peptide, resulted in loss of the ability of gE to bind IDE. A synthetic peptide corresponding to amino acids 24 to 50 of gE blocked its interaction with IDE in a concentration-dependent manner. However, a chimeric gE in which amino acids 1 to 71 of VZV gE were fused to amino acids 30 to 545 of herpes simplex virus type 2 gE did not show an increased level of binding to IDE compared with that of full-length HSV gE. Thus, amino acids 24 to 71 of gE are required for IDE binding, and the secondary structure of gE is critical for the interaction. VZV gE also forms a heterodimer with glycoprotein gI. Deletion of amino acids 163 to 208 of gE severely reduced its ability to form a complex with gI. The amino portion of IDE, as well an IDE mutant in the catalytic domain of the protein, bound to gE. Therefore, distinct motifs of VZV gE are important for binding to IDE or to gI.

Structural insights into dihydroxylation of terephthalate, a product of polyethylene terephthalate degradation

2022 ◽  
Author(s):  
Jai Krishna Mahto ◽  
Neetu Neetu ◽  
Monica Sharma ◽  
Monika Dubey ◽  
Bhanu Prakash Vellanki ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Substrate Specificity ◽  
Polyethylene Terephthalate ◽  
Active Site ◽  
Catalytic Domain ◽  
Structural Features ◽  
Comamonas Testosteroni ◽  
Plastic Pollution ◽  
Microbial Utilization ◽  
Steady State Kinetics

Biodegradation of terephthalate (TPA) is a highly desired catabolic process for the bacterial utilization of this Polyethylene terephthalate (PET) depolymerization product, but to date, the structure of terephthalate dioxygenase (TPDO), a Rieske oxygenase (RO) that catalyzes the dihydroxylation of TPA to a cis -diol is unavailable. In this study, we characterized the steady-state kinetics and first crystal structure of TPDO from Comamonas testosteroni KF1 (TPDO KF1 ). The TPDO KF1 exhibited the substrate specificity for TPA ( k cat / K m = 57 ± 9 mM −1 s −1 ). The TPDO KF1 structure harbors characteristics RO features as well as a unique catalytic domain that rationalizes the enzyme’s function. The docking and mutagenesis studies reveal that its substrate specificity to TPA is mediated by Arg309 and Arg390 residues, two residues positioned on opposite faces of the active site. Additionally, residue Gln300 is also proven to be crucial for the activity, its substitution to alanine decreases the activity ( k cat ) by 80%. Together, this study delineates the structural features that dictate the substrate recognition and specificity of TPDO. Importance The global plastic pollution has become the most pressing environmental issue. Recent studies on enzymes depolymerizing polyethylene terephthalate plastic into terephthalate (TPA) show some potential in tackling this. Microbial utilization of this released product, TPA is an emerging and promising strategy for waste-to-value creation. Research from the last decade has discovered terephthalate dioxygenase (TPDO), as being responsible for initiating the enzymatic degradation of TPA in a few Gram-negative and Gram-positive bacteria. Here, we have determined the crystal structure of TPDO from Comamonas testosteroni KF1 and revealed that it possesses a unique catalytic domain featuring two basic residues in the active site to recognize TPA. Biochemical and mutagenesis studies demonstrated the crucial residues responsible for the substrate specificity of this enzyme.

scholarly journals A novel industrial control architecture based on Software-Defined Network

2018 ◽  
Vol 51 (7-8) ◽  
pp. 360-367
Author(s):  
Geng Liang ◽  
Wen Li
Keyword(s):  
Large Scale ◽  
Service Management ◽  
Virtual Machines ◽  
Industrial Process ◽  
Control Network ◽  
Software Defined Network ◽  
Intelligent Network ◽  
Industrial Control ◽  
Control Functions ◽  
And Performance

Traditionally, routers and other network devices encompass both data and control functions in most large enterprise networks, making it difficult to adjust the network infrastructure and operation to large-scale addition of end systems, virtual machines, and virtual networks in industrial comprehensive automation. A network organizing technique that has come to recent prominence is the Software-Defined Network (SDN). A novel SDN based industrial control network (SDNICN) was proposed in this paper. Intelligent network components are included in a SDNICN. Switches in SDNICN provided fundamental network interconnection for the whole industrial control network. Network controller is used for data transmission, forwarding and routing control between different layers. Service Management Center (SMC) is essentially responsible for managing various services used in industrial process control. SDNICN can not only greatly improve the flexibility and performance of industrial control network but also meet the intelligence and informatization of the future industry.

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