Cysteine dioxygenase

Yamagata, Kenji; Hosokawa, Yu

doi:10.1016/0076-6879(87)43069-3

Cited by 40 publications

(30 citation statements)

References 17 publications

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“…This indicates that modification of Cys-90 is not because of nonspecific oxidation by atmospheric O 2 . Oxidation of a cysteine to cysteic sulfonic acid or sulfinic acid has previously been observed in a serine/threonine phosphatase (14), a nitrile hydratase (15), and the reaction catalyzed by cysteine dioxygenase (16). All three enzymes are iron metalloenzymes.…”

Section: Resultsmentioning

confidence: 99%

Oxygen-mediated Inactivation of Peptide Deformylase

Rajagopalan

Pei

1998

Journal of Biological Chemistry

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Peptide deformylase catalyzes the removal of the Nformyl group from newly synthesized polypeptides in prokaryotes. Its essential character and unique presence in prokaryotes make it an attractive target for antibacterial chemotherapy. However, purification and characterization of the peptide deformylase have remained a major challenge because this enzyme is extraordinarily labile under a variety of conditions (t 1/2 ϳ1 min at room temperature Protein synthesis in prokaryotes initiates with an N-formylmethionyl-tRNA i , resulting in N-terminal formylation of all nascent polypeptides (1). Peptide deformylase catalyzes the subsequent removal of the formyl group from the majority of bacterial proteins (2)(3)(4). Although the precise functions of the formylation and deformylation steps remain somewhat obscure, genetic studies have shown that the peptide deformylase activity is essential for bacterial survival (5, 6). Because peptide deformylase is apparently absent in eukaryotic systems (7,8), it provides a potential target for a novel antibacterial strategy (5). Very recently, biochemical studies from this laboratory have established that peptide deformylase represents an intriguing new class of amide hydrolyase, which utilizes a ferrous ion as the catalytic metal (7). The unusual structural feature of peptide deformylase and its potential application in antibiotic studies warrant a detailed mechanistic investigation of this class of enzymes.Although the deformylase activity was recognized three decades ago (2-4), the extraordinary lability of this enzyme has long prevented its purification and characterization. It was reported that even mild procedures such as dialysis, Sephadex G-100 or DEAE-cellulose chromatography, or ultracentrifugation on sucrose gradient resulted in almost quantitative loss of enzymatic activity (2, 3). At 37°C, the deformylase in a crude cell lysate lost activity with a half-life of 60 s (3). We have recently overexpressed the deformylase from Escherichia coli and purified the active enzyme to homogeneity (7, 9). Unfortunately, the purified enzyme remains highly unstable, complicating the quantitative evaluation of its activity. In this work, we have found that the instability of peptide deformylase is because of oxidation of the catalytic Fe 2ϩ ion into the catalytically inactive ferric ion by atmospheric O 2 . A simple experimental procedure has been developed to preserve the deformylase activity. These findings will greatly facilitate future mechanistic investigations of the peptide deformylase as well as high throughput screening for deformylase inhibitors. EXPERIMENTAL PROCEDURESMaterials-Glucose oxidase (EC 1.1.3.4), superoxide dismutase, and catalase (EC 1.11.1.6) from Aspergillus niger were purchased from Fluka (Ronkonkoma, NY). Aeromonas aminopeptidase and all chemicals were from Sigma Chemical Company.Buffers-All purification buffers were exhaustively degassed under vacuum and then sparged with argon prior to use. Buffer A, 20 mM potassium phosphate, pH 8.0, 10 mM NaCl, 1% Triton X...

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Section: Resultsmentioning

confidence: 99%

Oxygen-mediated Inactivation of Peptide Deformylase

Rajagopalan

Pei

1998

Journal of Biological Chemistry

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“…The rat liver CDO purified by Yamaguchi and Hosokawa (24) was characterized as a single subunit protein composed of 200 amino acid residues with a molecular mass of 22.5 kDa and a pI value of 5.5. Atomic absorption analysis indicated 0.8 moles of iron per mole of enzyme.…”

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confidence: 99%

Heterologous Expression, Purification, and Characterization of Recombinant Rat Cysteine Dioxygenase

Chai

Jerkins

Banik

et al. 2005

Journal of Biological Chemistry

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Cysteine dioxygenase (CDO, EC 1.13.11.20) catalyzes the oxidation of cysteine to cysteine sulfinic acid, which is the first major step in cysteine catabolism in mammalian tissues. Rat liver CDO was cloned and expressed in Escherichia coli as a 26.8-kDa N-terminal fusion protein bearing a polyhistidine tag. Purification by immobilized metal affinity chromatography yielded homogeneous protein, which was catalytically active even in the absence of the secondary protein-A, which has been reported to be essential for activity in partially purified native preparations. As compared with those existing purification protocols for native CDO, the milder conditions used in the isolation of the recombinant CDO allowed a more controlled study of the properties and activity of CDO, clarifying conflicting findings in the literature. Apo-protein was inactive in catalysis and was only activated by iron. Metal analysis of purified recombinant protein indicated that only 10% of the protein contained iron and that the iron was loosely bound to the protein. Kinetic studies showed that the recombinant enzyme displayed a K m value of 2.5 ؎ 0.4 mM at pH 7.5 and 37°C. The enzyme was shown to be specific for L-cysteine oxidation, whereas homocysteine inhibited CDO activity.

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“…The supernatant obtained was used as a crude enzyme extract. The CDO activity was measured radiochemically by the method of Yamaguchi and Hosokawa (1987a) using L-[35S]cysteine as a substrate in the presence of a sufficient amount of CDO-stabilizing protein prepared from a rat liver extract (Yamaguchi and Hosokawa 1987b;Yokoyama and Nakazoe 1989). The assay mixture contained 0.3 ml of a crude enzyme extract, 10 tamol of L-[35S]cysteine (pH 6.8), I00 lamol of glycineNaOH buffer (pH 9.0) and stabilizing protein in the final volume of 1 ml.…”

Section: Assay Of Hepatic Cdo Activi~mentioning

confidence: 99%

“…However, little is known about the sulfur amino acid metabolism in fish, though the fish tissues generally contain a large amount of taurine (Sakaguchi and Murata 1988). It is accepted that L-cysteinesulfinate is a key intermediate product both in the catabolic pathway to form pyruvate and sulfate, and in taurine biosynthesis pathway from L-cysteine in mammals as shown in Figure 1 (Yamaguchi and Hosokawa 1987a;Yamaguchi 1990). Therefore, cysteine dioxygenase (CDO) [EC 1.13.11.20] which catalyzes the oxygenation of L-cysteine to form Lcysteine sulfinate has been considered to participate in the regulation of sulfur amino acid metabolism in mammals (Daniels and Stipanuk 1982;Hosokawa et al 1988).…”

Section: Introductionmentioning

confidence: 99%

Intraperitoneal injection of sulfur amino acids enhance the hepatic cysteine dioxygenase activity in rainbow trout (Oncorhynchus mykiss)

Yokoyama

Nakazoe

1996

Fish Physiol Biochem

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As part of the investigation into cysteine metabolism in fish, sulfur amino acids and their derivatives were injected intraperitoneally to fingerling rainbow trout (Oncorhynchus mykiss) to examine how the doses of these compounds affect the hepatic cysteine dioxygenase [EC 1.12.11.20] in this species. A dose of 0.25 mmol L-cysteine per 100 g body weight induced the enzyme activity as much as 2.5 times that of the control fish within 4h after the injection. The activity increased proportionally to the increasing dose of cysteine up to the dose of 0.15 mmol per 100 g body weight. The induction was observed to be rather specific to Lcysteine. These findings suggested that the cysteine sulfinate pathway might play an important role in the metabolism of excess cysteine in rainbow trout. The dosage of L-cysteine larger than 0.50 mmol per 100g body weight led to mortality of the fish. The pathway of cysteine catabolism was considered to function to prevent toxic accumulation of cysteine in rainbow trout, as in the case of mammals.

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Cysteine dioxygenase

Cited by 40 publications

References 17 publications

Oxygen-mediated Inactivation of Peptide Deformylase

Oxygen-mediated Inactivation of Peptide Deformylase

Heterologous Expression, Purification, and Characterization of Recombinant Rat Cysteine Dioxygenase

Intraperitoneal injection of sulfur amino acids enhance the hepatic cysteine dioxygenase activity in rainbow trout (Oncorhynchus mykiss)

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