Stoichiometry of the pyrimidine deoxyribonucleoside 2'-hydroxylase reaction and of the conversions of 5-hydroxymethyluracil to 5-formyluracil and of the latter to uracil-5-carboxylic acid

Liu, Chen Kao; Shaffer, Patricia M.; Slaughter, Robert S.; McCroskey, R.P.; Abbott, Mitchel T.

doi:10.1021/bi00761a025

Cited by 24 publications

(14 citation statements)

References 13 publications

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“…Recently, the fungal thymidine salvage pathway has attracted great attention due to the similarity in the chemistries of the T-to-U and the 5mC-to-C conversions [8,12,15] (Supplementary information, Figure S1). In fungi, the demethylation of T to U is a multistep process including the conversion of T to 5hmU, 5fU and 5caU through three consecutive oxidation reactions catalyzed by T7H and the non-oxidative decarboxylation of 5caU to U catalyzed by IDCase [22][23][24][25][26]. In mammals, the TET proteins can sequentially oxidize 5mC to 5hmC, 5fC and 5caC [9][10][11][12].…”

Section: Catalytic Mechanism Of Decarboxylation For Idcasesmentioning

confidence: 99%

“…However, Neurospora crassa and several other fungi can demethylate T to U via a unique thymidine salvage pathway [21]. This pathway consists of a series of catalytic reactions, including the oxidation of thymidine to thymine ribonucleoside by pyrimidine deoxyribonucleoside 2′-hydroxylase, the hydrolysis of thymine ribonucleoside to thymine and ribose by pyrimidine-nucleoside phosphorylase and hydrolase, the sequential oxidation of thymine to 5-hydroxymethyl-uracil (5hmU), 5-formyluracil (5fU) and 5-carboxyl-uracil (5caU) by thymine-7-hydroxylase (T7H) [22][23][24][25][26], and finally the non-oxidative decarboxylation of 5caU to U by isoorotate decarboxylase (IDCase) [27,28].…”

Section: Introductionmentioning

confidence: 99%

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Crystal structures of isoorotate decarboxylases reveal a novel catalytic mechanism of 5-carboxyl-uracil decarboxylation and shed light on the search for DNA decarboxylase

Zhu

et al. 2013

Cell Res

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DNA methylation and demethylation regulate many crucial biological processes in mammals and are linked to many diseases. Active DNA demethylation is believed to be catalyzed by TET proteins and a putative DNA decarboxylase that may share some similarities in sequence, structure and catalytic mechanism with isoorotate decarboxylase (IDCase) that catalyzes decarboxylation of 5caU to U in fungi. We report here the structures of wild-type and mutant IDCases from Cordyceps militaris and Metarhizium anisopliae in apo form or in complexes with 5caU, U, and an inhibitor 5-nitro-uracil. IDCases adopt a typical (β/α) 8 barrel fold of the amidohydrolase superfamily and function as dimers. A Zn 2+ is bound at the active site and coordinated by four strictly conserved residues, one Asp and three His. The substrate is recognized by several strictly conserved residues. The functional roles of the key residues at the active site are validated by mutagenesis and biochemical studies. Based on the structural and biochemical data, we present for the first time a novel catalytic mechanism of decarboxylation for IDCases, which might also apply to other members of the amidohydrolase superfamily. In addition, our biochemical data show that IDCases can catalyze decarboxylation of 5caC to C albeit with weak activity, which is the first in vitro evidence for direct decarboxylation of 5caC to C by an enzyme. These findings are valuable in the identification of potential DNA decarboxylase in mammals.

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Section: Catalytic Mechanism Of Decarboxylation For Idcasesmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Crystal structures of isoorotate decarboxylases reveal a novel catalytic mechanism of 5-carboxyl-uracil decarboxylation and shed light on the search for DNA decarboxylase

Zhu

et al. 2013

Cell Res

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“…In the 1970s, Shaffer et al separated two enzymes, thymine-7-hydroxylase (THase; official name thymine dioxygenase) and isoorotate decarboxylase (IDCase), from fungal sources which showed potential ability to convert thymine to uracil (22). THase is a trifunctional oxygenase and catalyzes three enzymatic reactions: thymine to 5-hydroxymethyluracil, 5-hydroxymethyluracil to 5-formyluracil, and 5-formyluracil to uracil-5-carboxylic acid (also designated isoorotate) (18,26). IDCase catalyzes the subsequent decarboxylation reaction, and uracil-5-carboxylic acid is converted to uracil in the end (21).…”

mentioning

confidence: 99%

Enhancement of the Diversity of Polyoxins by a Thymine-7-Hydroxylase Homolog outside the Polyoxin Biosynthesis Gene Cluster

Zhao

Huang

Chen

et al. 2010

Appl Environ Microbiol

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“…The sources of the radioactive comp described previously (19,22,26). The sp5 ties (curies per mole) of the compounds follows: 3.0 for [2-14C]thymine, 2.0 for [7-' carboxylic acid, and 0.50 for a-[1-'4C]kel All other fine chemicals were obtained mercial sources as previously described Growth of mycelium for enzymatic nidia were obtained from cultures that for 10 days (at 28 C in constant light) Fries minimal medium, solidified with 125-ml Erlenmeyer flasks.…”

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confidence: 99%

“…The assays used for the cell-free studies were based on the rate of formation of radioactive C02 from carboxy-labeled a-ketoglutarate. The standard incubation mixture (0.225 ml) was 10 mM in sodium phosphate, pH 7.5, and contained 0.1 ml of the enzyme preparation, 1 mM a-_1-14C]ketoglutarate, 1 mM ferrous sulfate, 1 mM ascorbate, 0.75 mM thymine or deoxyuridine, and 0.4 mg of catalase per ml (19,22,26). Since crude extracts could decarboxylate a-ketoglutarate in the absence of exogenous thymine and deoxyuridine, corrections were made in the calculation of enzyme activities with the values obtained from such control incubations.…”

mentioning

confidence: 99%

Regulation of thymidine metabolism in Neurospora crassa

Griswold¹,

Madrid²,

Shaffer³

et al. 1976

J Bacteriol

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The utilization of thymidine by Neurospora crassa is initiated by the pyrimidine deoxyribonucleoside 2'-hydroxylase reaction and the consequent formation of thymine and ribose. Thymine must then be oxidatively demethylated by the thymine 7-hydroxylase and uracil-5-carboxylic acid decarboxylase reactions. This article shows that the 2'-hydroxylase reaction can be regulated differently than the oxidative demethylation process and suggests that the 2'-hydroxylase has, in addition to the role of salvaging the pyrimidine ring, the role of providing ribose not only for the utilization of the demethylated pyrimidine but also for other metabolic processes. One way that this difference in regulation was observed was with the uc-1 mutation developed by Williams and Mitchell. The present communication shows that this mutation increases the activities of the 7-hydroxylase and the decarboxylase but has no comparable effect on the 2'hydroxylase. Qualitatively similar effects on these enzymes were brought about by growth of wild-type Neurospora in media lacking ammonium ion, such as the Westergaard-Mitchell medium. The 2'-hydroxylase and 7-hydroxylase are also differently affected by the carbon dioxide content of the atmosphere above the growing culture and the growth temperature. Studies with inhibitors indicated that the carbon dioxide effect is dependent on protein synthesis. Recent studies (34) have indicated that in order for Neurospora crassa to utilize pyrimidine deoxyribonucleosides they must be initially metabolized as depicted in Fig. 1. This report describes several factors that may be pertinent to the regulation of the pathway. The first indication of one ofthese factors came from attempts to facilitate purification of thymine 7hydroxylase by growing liquid cultures under forced aeration. Although the yield of mycelia was increased over that obtained from shallow liquid cultures, the 7-hydroxylase activity was markedly decreased and, furthermore, the incorporation of thymidine into ribonucleic acid was inhibited (22). However, larger nonaerated cultures with considerable 7-hydroxylase activity could be obtained if the Neurospora were grown with stirring in a sealed carboy halffilled with medium (22). Since the atmosphere inside of the carboy became anaerobic during growth, it was suspected that an increase in the 7-hydroxylase activity might, somehow, be initiated by this change in the gas composition.Another factor that may play a regulatory role is the ammonium ion content of the growth medium. The Westergaard-Mitchell medium permits the efficient utilization of intermediates in the pathway shown in Fig. 1 (39). The data in the present article indicate that mycelia grown in this medium contain higher activities

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Stoichiometry of the pyrimidine deoxyribonucleoside 2'-hydroxylase reaction and of the conversions of 5-hydroxymethyluracil to 5-formyluracil and of the latter to uracil-5-carboxylic acid

Cited by 24 publications

References 13 publications

Crystal structures of isoorotate decarboxylases reveal a novel catalytic mechanism of 5-carboxyl-uracil decarboxylation and shed light on the search for DNA decarboxylase

Crystal structures of isoorotate decarboxylases reveal a novel catalytic mechanism of 5-carboxyl-uracil decarboxylation and shed light on the search for DNA decarboxylase

Enhancement of the Diversity of Polyoxins by a Thymine-7-Hydroxylase Homolog outside the Polyoxin Biosynthesis Gene Cluster

Regulation of thymidine metabolism in Neurospora crassa

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