Oxygenases involved in thymine and thymidine metabolism in<i>Neurospora crassa</i>

Bankel, Louise; Holme, Elisabeth; Lindstedt, Göran; Lindstedt, Sven

doi:10.1016/0014-5793(72)80121-2

Cited by 23 publications

(10 citation statements)

References 11 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Expandase activity required 2-oxoglutarate, Fe2+ ions and 02 for activity, with stimulation by ascorbate and DTT. These are similar requirements to those exhibited by the oxygenases thymidine 2-hydroxylase (Bankel et al, 1972) and collagen hydroxylases (Majamaa et al, 1985), in which the 2-oxoglutarate is stoichiometrically decarboxylated to succinate. In the fungus Neurospora crassa thymine 7-hydroxylation is accomplished by a 2-oxoglutarate-requiring system that involves three discrete steps, all of which appear to be associated with a single enzyme (Liu et al, 1973 assay system with succinate thiokinase, pyruvate kinase and lactate dehydrogenase, we have shown that both expandase and hydroxylase activities produce succinate from 2-oxoglutarate stoichiometrically with the production of cephem (Baldwin & Crabbe, 1987).…”

Section: Discussionsupporting

confidence: 64%

Purification and initial characterization of an enzyme with deacetoxycephalosporin C synthetase and hydroxylase activities

Baldwin

Adlington

Coates

et al. 1987

Biochemical Journal

View full text Add to dashboard Cite

Deacetoxycephalosporin C synthetase (expandase) from Cephalosporium acremonium (Acremonium chrysogenum) was purified to near homogeneity as judged by SDS/polyacrylamide-gel electrophoresis. The enzyme (Mr about 40,000) exhibited a pH optimum around 7.5. It required 2-oxoglutarate (Km 0.04 mM), Fe2+ and O2 as cofactors, and ascorbate and dithiothreitol were necessary for maximum activity. It was stable for over 4 weeks at -70 degrees C in the presence of 1 mM-dithiothreitol. Activity was inhibited by the thiol-quenching reagent N-ethylmaleimide, the metal-ion-chelating reagent bathophenanthroline, and NH4HCO3. The highly purified enzyme also showed deacetoxycephalosporin C hydroxylase (deacetylcephalosporin C synthetase) activity, indicating that both expandase and hydroxylase activities are properties of a single protein. These activities could not be separated by ion-exchange, dye-ligand, gel-filtration or hydrophobic chromatography. A beta-sulphoxide and a 3 beta-methylene hydroxy analogue of penicillin N were synthesized to test as potential intermediates in the ring-expansion reaction, Neither compound was a substrate for the enzyme. A synthetic analogue in which the 3 beta-methyl group and the 2-hydrogen atom of penicillin N were replaced by a cyclopropane ring was not a substrate but was a reversible inhibitor of the enzyme.

show abstract

Section: Discussionsupporting

confidence: 64%

Purification and initial characterization of an enzyme with deacetoxycephalosporin C synthetase and hydroxylase activities

Baldwin

Adlington

Coates

et al. 1987

Biochemical Journal

View full text Add to dashboard Cite

show abstract

“…Another iron/αKG-dependent dioxygenase is the fungal enzyme thymidine 2′-hydroxylase (pyrimidine-deoxynucleoside 2′-dioxygenase) from Neurospora crassa which lost ≥95% of its in vitro ability to hydroxylate pyrimidines upon exposure to 250 μM Ni(II). 97 Inhibition of the Fe/αKG-dependent dioxygenases in microorganisms parallels the findings in mammalian systems where nickel inhibits histone demethylases (JMJD1A, JMJD2, and JMJD2E), oxygen-sensing prolyl 4-hydroxylases (PHD2, EGLN1), and DNA repair nucleotide demethylases (ABH2 and ABH3) by replacement of the catalytic iron atom. 85–87 Inhibition of these chromatin modification enzymes may explain the nickel-dependent mutagenic and epigenetic phenotypes observed in mammals.…”

Section: 0 Nickel Toxicitymentioning

confidence: 67%

Mechanisms of nickel toxicity in microorganisms

2011

View full text Add to dashboard Cite

Summary Nickel has long been known to be an important human toxicant, including having the ability to form carcinomas, but until recently nickel was believed to be an issue only to microorganisms living in nickel-rich serpentine soils or areas contaminated by industrial pollution. This assumption was overturned by the discovery of a nickel defense system (RcnR/RcnA) found in microorganisms that live in a wide range of environmental niches, suggesting that nickel homeostasis is a general biological concern. To date, the mechanisms of nickel toxicity in microorganisms and higher eukaryotes are poorly understood. In this review, we summarize nickel homeostasis processes used by microorganisms and highlight in vivo and in vitro effects of exposure to elevated concentrations of nickel. On the basis of this evidence we propose four mechanisms of nickel toxicity: 1) nickel replaces the essential metal of metalloproteins, 2) nickel binds to catalytic residues of non-metalloenzymes; 3) nickel binds outside the catalytic site of an enzyme to inhibit allosterically, and 4) nickel indirectly causes oxidative stress.

show abstract

“…They are involved in a broad spectrum of primary and secondary biosynthetic pathways including the post-translational hydroxylation of proline and lysine residues in procollagens (Kivirikko et al, 1989), the biosynthesis of carnitine ) and blood-coagulation-factor VII (Stenflo et al, 1989) in mammals, the conversion of thymidine into uracil (Holme et al, 1970(Holme et al, , 1971Bankel et al, 1977) and of penicillins into cephalosporins (Baldwin and Abraham, 1988) in bacteria and fungi, the biosynthesis of clavulanic acid (Elson et al, 1987) and the macrolide antibioticum tylosin (Omura et al, 1984) in fungi, the formation of hydroxyproline-rich glycoproteins in plants (Chrispeels, 1969;Tanaka et al, 1980) and the biosynthesis of plant secondary products such a5 flavonoids Britsch et al, 1981), the alkaloids scopolamine (Hashimot0 and Yamada, 1986) and vindoline (De Carolis et al, 1990) and gibberellins (Hedden and Graebe, 1982).…”

Section: Discussionmentioning

confidence: 99%

Molecular characterization of flayanone 3β‐hydroxylases

Britsch

Dedio²,

Saedler³

et al. 1993

European Journal of Biochemistry

101

View full text Add to dashboard Cite

A heterologous cDNA probe from Petunia hybrida was used to isolate flavanone-3P-hydroxylase-encoding cDNA clones from carnation (Dianthus caryophyllus), china aster (Callisteplzus chinensis) and stock (Matthiola incana). The deduced protein sequences together with the known sequences of the enzyme from l ? hybrida, barley (Hordeum vulgare) and snapdragon (Antirrhinum majus) enabled the determination of a consensus sequence which revealed an overall 84% similarity (53 % identity) of flavanone 3P-hydroxylases from the different sources. Alignment with the sequences of other known enzymes of the same class and to related non-heme iron-(11) enzymes demonstrated the strict genetic conservation of 14 anuno acids, in particular, of three histidines and an aspartic acid. The conservation of the histidine motifs provides strong support for the possible conservation of structurally similar iron-binding sites in these enzymes. The putative role of histidines as chelators of ferrous ions in the active site of tlavanone 3P-hydroxylases was corroborated by diethyl-pyrocarbonate modification of the partially purified recombinant Petunia enzyme.Flavanone 3P-hydroxylase (FHT) catalyzes 3P-hydroxylation of 2S-flavanones to 2R,3R-dihydroflavonols [( +)-dihydroflavonols] which are intermediates in the biosynthesis of flavonols, anthocyanidins, catechins and proanthocyanidins in plants (Heller and Forkmann, 1988;Forkmann, 1991). The enzyme from Petunia hybrida has been purified to homogeneity and characterized as a typical 2-oxoglutarate-dependent dioxygenase (Britsch and Grisebach, 1986;Britsch, 1990). Recently, we have isolated a near-full-length FHTencoding cDNA from a Petunia library (Britsch et al., 1992). Heterologous expression of the cDNA in Escherichia coli led to the formation of highly active recombinant FHT, thereby verifying the identity of the cDNA clone.Antibodies against FHT from Petunia readily crossreact with FHT from a number of other sources and were used to characterize specific FHT mutants (Britsch, 1990; Koch, 1992). Therefore, structural similarities of the FHT protein from different plants can be assumed. A comparison of the deduced amino acid sequence of the Petunia FHT with the sequence of FHT from barley (Meldgaard, 1992) indeed showed a high degree of identity (75%; Britsch et al., 1992).Consequently, the FHT cDNA from Petunia should serve as a useful heterologous probe to clone this enzyme from other plant sources. Utilizing the 945-bp EcoRI fragment from Petunia FHT as a hybridization probe, we were able to clone the corresponding cDNAs from carnation, china aster and stock. In this study, we report the cloning and sequence analysis of these particular FHT enzymes, the analysis of the consensus sequence derived from six individual FHT sequences and the modification of purified recombinant enzyme with diethyl pyrocarbonate, performed to elucidate the possible function of strictly conserved histidines. Furthermore, the sequences were also compared with other known 2-oxoglutarate-dependent dioxygenases f...

show abstract

Oxygenases involved in thymine and thymidine metabolism inNeurospora crassa

Cited by 23 publications

References 11 publications

Purification and initial characterization of an enzyme with deacetoxycephalosporin C synthetase and hydroxylase activities

Purification and initial characterization of an enzyme with deacetoxycephalosporin C synthetase and hydroxylase activities

Mechanisms of nickel toxicity in microorganisms

Molecular characterization of flayanone 3β‐hydroxylases

Contact Info

Product

Resources

About