WOR5, a Novel Tungsten-Containing Aldehyde Oxidoreductase from Pyrococcus furiosus with a Broad Substrate Specificity

ABSTRACT WOR5 is the fifth and last member of the family of tungsten-containing oxidoreductases purified from the hyperthermophilic archaeon Pyrococcus furiosus. It is a homodimeric protein (subunit, 65 kDa) that contains one [4Fe-4S] cluster and one tungstobispterin cofactor per subunit. It has a broad substrate specificity with a high affinity for several substituted and nonsubstituted aliphatic and aromatic aldehydes with various chain lengths. The highest catalytic efficiency of WOR5 is found for the oxidation of hexanal (V max = 15.6 U/mg, Km = 0.18 mM at 60°C). Hexanal-incubated enzyme exhibits S = 1/2 electron paramagnetic resonance signals from [4Fe-4S]1+ (g values of 2.08, 1.93, and 1.87) and W5+ (g values of 1.977, 1.906, and 1.855). Cyclic voltammetry of ferredoxin and WOR5 on an activated glassy carbon electrode shows a catalytic wave upon addition of hexanal, suggesting that ferredoxin can be a physiological redox partner. The combination of WOR5, formaldehyde oxidoreductase, and aldehyde oxidoreductase forms an efficient catalyst for the oxidation of a broad range of aldehydes in P. furiosus.

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Molybdenum Incorporation in Tungsten Aldehyde Oxidoreductase Enzymes from Pyrococcus furiosus

Journal of Bacteriology ◽

10.1128/jb.00270-10 ◽

2010 ◽

Vol 192 (16) ◽

pp. 4143-4152 ◽

Cited By ~ 30

Author(s):

Ana-Maria Sevcenco ◽

Loes E. Bevers ◽

Martijn W. H. Pinkse ◽

Gerard C. Krijger ◽

Hubert T. Wolterbeek ◽

...

Keyword(s):

Pyrococcus Furiosus ◽

Growth Conditions ◽

Oxidation Activity ◽

Paramagnetic Resonance ◽

Aldehyde Oxidoreductase ◽

Associated Proteins ◽

Main Carbon Source ◽

Oxidoreductase Enzymes ◽

Aldehyde Oxidation ◽

Electron Paramagnetic

ABSTRACT The hyperthermophilic archaeon Pyrococcus furiosus expresses five aldehyde oxidoreductase (AOR) enzymes, all containing a tungsto-bispterin cofactor. The growth of this organism is fully dependent on the presence of tungsten in the growth medium. Previous studies have suggested that molybdenum is not incorporated in the active site of these enzymes. Application of the radioisotope 99Mo in metal isotope native radioautography in gel electrophoresis (MIRAGE) technology to P. furiosus shows that molybdenum can in fact be incorporated in all five AOR enzymes. Mo(V) signals characteristic for molybdopterin were observed in formaldehyde oxidoreductase (FOR) in electron paramagnetic resonance (EPR)-monitored redox titrations. Our finding that the aldehyde oxidation activity of FOR and WOR5 (W-containing oxidoreductase 5) correlates only with the residual tungsten content suggests that the Mo-containing AORs are most likely inactive. An observed W/Mo antagonism is indicative of tungstate-dependent negative feedback of the expression of the tungstate/molybdate ABC transporter. An intracellular selection mechanism for tungstate and molybdate processing has to be present, since tungsten was found to be preferentially incorporated into the AORs even under conditions with comparable intracellular concentrations of tungstate and molybdate. Under the employed growth conditions of starch as the main carbon source in a rich medium, no tungsten- and/or molybdenum-associated proteins are detected in P. furiosus other than the high-affinity transporter, the proteins of the metallopterin insertion machinery, and the five W-AORs.

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Substrate specificity and structure of human aminoadipate aminotransferase/kynurenine aminotransferase II

Bioscience Reports ◽

10.1042/bsr20080085 ◽

2008 ◽

Vol 28 (4) ◽

pp. 205-215 ◽

Cited By ~ 57

Author(s):

Qian Han ◽

Tao Cai ◽

Danilo A. Tagle ◽

Howard Robinson ◽

Jianyong Li

Keyword(s):

Substrate Specificity ◽

Catalytic Efficiency ◽

Structural Basis ◽

Substrate Affinity ◽

Kinetic Properties ◽

Amino Group ◽

Kynurenine Aminotransferase ◽

Broad Substrate Specificity ◽

Systematic Assessment ◽

The Brain

KAT (kynurenine aminotransferase) II is a primary enzyme in the brain for catalysing the transamination of kynurenine to KYNA (kynurenic acid). KYNA is the only known endogenous antagonist of the N-methyl-D-aspartate receptor. The enzyme also catalyses the transamination of aminoadipate to α-oxoadipate; therefore it was initially named AADAT (aminoadipate aminotransferase). As an endotoxin, aminoadipate influences various elements of glutamatergic neurotransmission and kills primary astrocytes in the brain. A number of studies dealing with the biochemical and functional characteristics of this enzyme exist in the literature, but a systematic assessment of KAT II addressing its substrate profile and kinetic properties has not been performed. The present study examines the biochemical and structural characterization of a human KAT II/AADAT. Substrate screening of human KAT II revealed that the enzyme has a very broad substrate specificity, is capable of catalysing the transamination of 16 out of 24 tested amino acids and could utilize all 16 tested α-oxo acids as amino-group acceptors. Kinetic analysis of human KAT II demonstrated its catalytic efficiency for individual amino-group donors and acceptors, providing information as to its preferred substrate affinity. Structural analysis of the human KAT II complex with α-oxoglutaric acid revealed a conformational change of an N-terminal fraction, residues 15–33, that is able to adapt to different substrate sizes, which provides a structural basis for its broad substrate specificity.

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Characterization of a Fourth Tungsten-Containing Enzyme from the Hyperthermophilic Archaeon Pyrococcus furiosus

Journal of Bacteriology ◽

10.1128/jb.184.24.6952-6956.2002 ◽

2002 ◽

Vol 184 (24) ◽

pp. 6952-6956 ◽

Cited By ~ 44

Author(s):

Roopali Roy ◽

Michael W. W. Adams

Keyword(s):

Pyrococcus Furiosus ◽

Carbon Sources ◽

Aromatic Aldehydes ◽

Optimal Growth ◽

Paramagnetic Resonance ◽

Cell Extracts ◽

Keto Acids ◽

Approximate Molecular Weight ◽

Electron Paramagnetic

ABSTRACT Pyrococcus furiosus grows optimally near 100°C using peptides and carbohydrates as carbon sources, and it reduces elemental sulfur (S0), if present, to H2S. Tungsten (W), an element rarely used in biology, is required for optimal growth, and three different tungsten-containing enzymes have been previously purified from this organism. They all oxidize aldehydes of various types and are thought to play primary roles in the catabolism of sugars or amino acids. Here, the purification of a fourth tungsten-containing enzyme, termed WOR 4, from cell extracts of P. furiosus grown with S0 is described. This was achieved by monitoring through multiple chromatography steps the W that is not associated with the three characterized tungstoenzymes. The N-terminal sequence of WOR 4 and the approximate molecular weight of its subunit determined electrophoretically (69,000) correspond to the product of an ORF (PF1961, wor4) present in the complete genome sequence of P. furiosus. WOR 4 is a homodimer and contains approximately one W, three Fe, three or four acid-labile sulfide, and one Ca atom per subunit. The visible and electron paramagnetic resonance spectra of the oxidized and reduced enzyme indicate the presence of an unusual iron-sulfur chromophore. WOR 4 does not oxidize aliphatic or aromatic aldehydes or hydroxy acids, nor does it reduce keto acids. Consistent with prior microarray data, the protein could not be purified from P. furiosus cells grown in the absence of S0, suggesting that it may have a role in S0 metabolism.

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Efficient Catalyst One-Pot Synthesis of 7-(Aryl)-10,10-dimethyl-10,11-dihydrochromeno[4,3-b]chromene-6,8(7H,9H)-dione Derivatives Complemented by Antibacterial Activity

BioMed Research International ◽

10.1155/2016/5891703 ◽

2016 ◽

Vol 2016 ◽

pp. 1-7 ◽

Cited By ~ 1

Author(s):

Yasameen K. Al-Majedy ◽

Ahmed A. Al-Amiery ◽

Abdul Amir H. Kadhum ◽

Abu Bakar Mohamad

Keyword(s):

Antibacterial Activity ◽

Tetrabutylammonium Bromide ◽

Reaction Times ◽

Catalytic Efficiency ◽

Aromatic Aldehydes ◽

One Pot ◽

Efficient Catalyst ◽

One Pot Synthesis ◽

Bacteria Resistance ◽

Diammonium Hydrogen

The problem of bacteria resistance to many known agents has inspired scientists and researchers to discover novel efficient antibacterial drugs. Three rapid, clean, and highly efficient methods were developed for one-pot synthesis of 7-(aryl)-10,10-dimethyl-10,11-dihydrochromeno[4,3-b]chromene-6,8(7H,9H)-dione derivatives. Three components are condensed in the synthesis, 4-hydroxycoumarin, 5,5-dimethyl-1,3-cyclohexanedione, and aromatic aldehydes, using tetrabutylammonium bromide (TBAB), diammonium hydrogen phosphate (DAHP), or ferric chloride (FeCl3), respectively. Each method has different reaction mechanisms according to the catalyst. The present methods have advantages, including one-pot synthesis, excellent yields, short reaction times, and easy isolation of product. All catalysts utilized in our study could be reused several times without losing their catalytic efficiency. All synthesized compounds were fully characterized and evaluated for their antibacterial activity.

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Faculty Opinions recommendation of UDP-sugar pyrophosphorylase with broad substrate specificity toward various monosaccharide 1-phosphates from pea sprouts.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1021628.246636 ◽

2004 ◽

Author(s):

Christoph Benning

Keyword(s):

Substrate Specificity ◽

Broad Substrate Specificity

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Faculty Opinions recommendation of A rice family 9 glycoside hydrolase isozyme with broad substrate specificity for hemicelluloses in type II cell walls.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.722918335.793513580 ◽

2016 ◽

Author(s):

Daniel Cosgrove

Keyword(s):

Substrate Specificity ◽

Cell Walls ◽

Glycoside Hydrolase ◽

Type Ii ◽

Broad Substrate Specificity ◽

Type Ii Cell

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SO3H-functionalized Zeolite-Y as an Efficient Nanocatalyst for the Synthesis of Nbenzimidazole- 2-aryl-4-thiazolidinones and tri-substituted Imidazoles

Current Organic Synthesis ◽

10.2174/1570179417666200115170019 ◽

2020 ◽

Vol 17 (2) ◽

pp. 117-130 ◽

Cited By ~ 1

Author(s):

Mehdi Kalhor ◽

Zohre Zarnegar ◽

Zahra Seyedzade ◽

Soodabeh Banibairami

Keyword(s):

Zeolite Y ◽

Internal Standard ◽

Acid Catalyst ◽

Aromatic Aldehydes ◽

Reaction Rates ◽

Ambient Conditions ◽

Catalytic Method ◽

Efficient Catalyst ◽

Bet Analysis ◽

Ft Ir

Background: SO3H-functionalized zeolite-Y was prepared and used as a catalyst for the synthesis of 2-aryl-N-benzimidazole-4-thiazolidinones and tri-substituted imidazoles at ambient conditions. Objective: The goals of this catalytic method include excellent yields and high purity, inexpensive procedure and ease of product isolation, the use of nontoxic and heterogeneous acid catalyst, shorter reaction times and milder conditions. Materials and Methods: NMR spectra were recorded on Brucker spectrophotometer using Me4Si as internal standard. Mass spectra were recorded on an Agilent Technology 5975C VL MSD with tripe-axis detector. FTIR spectra were obtained with KBr disc on a galaxy series FT-IR 5000 spectrometer. The surface morphology of nanostructures was analyzed by FE-SEM (EVO LS 10, Zeiss, Carl Zeiss, Germany). BET analysis were measured at 196 °C by a Japan Belsorb II system after the samples were vacuum dried at 150°C overnight. Results: The NSZ was characterized by FT-IR, FESEM, EDX, XRF, and BET. The catalytic activity of NSZ was investigated for synthesis of 1,3-tiazolidin-4-ones in H2O/Acetone at room temperature. Moreover, NSZ was used for synthesis of tri-substituted imidazoles at 60 °C via solvent-free condensation. Different kinds of aromatic aldehydes were converted to the corresponding of products with good to excellent yields. Conclusion: Sulfonated zeolite-Y was as an efficient catalyst for the preparation of N-benzimidazole-2-aryl-1,3- thiazolidin-4-ones and 2,4,5-triaryl-1H-imidazoles. High reaction rates, elimination toxic solvent, simple experimental procedure and reusability of the catalyst are the important features of this protocol.

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LASS3 (longevity assurance homologue 3) is a mainly testis-specific (dihydro)ceramide synthase with relatively broad substrate specificity

Biochemical Journal ◽

10.1042/bj20060379 ◽

2006 ◽

Vol 398 (3) ◽

pp. 531-538 ◽

Cited By ~ 116

Author(s):

Yukiko Mizutani ◽

Akio Kihara ◽

Yasuyuki Igarashi

Keyword(s):

Amino Acid ◽

Substrate Specificity ◽

Family Members ◽

Fatty Acyl ◽

Ceramide Synthase ◽

Broad Substrate Specificity ◽

Acid Protein ◽

Amino Acid Protein

The LASS (longevity assurance homologue) family members are highly conserved from yeasts to mammals. Five mouse and human LASS family members, namely LASS1, LASS2, LASS4, LASS5 and LASS6, have been identified and characterized. In the present study we cloned two transcriptional variants of hitherto-uncharacterized mouse LASS3 cDNA, which encode a 384-amino-acid protein (LASS3) and a 419-amino-acid protein (LASS3-long). In vivo, [3H]dihydrosphingosine labelling and electrospray-ionization MS revealed that overproduction of either LASS3 isoform results in increases in several ceramide species, with some preference toward those having middle- to long-chain-fatty acyl-CoAs. A similar substrate preference was observed in an in vitro (dihydro)ceramide synthase assay. These results indicate that LASS3 possesses (dihydro)ceramide synthesis activity with relatively broad substrate specificity. We also found that, except for a weak display in skin, LASS3 mRNA expression is limited almost solely to testis, implying that LASS3 plays an important role in this gland.

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