Bovine liver dihydropyrimidine amidohydrolase: Purification, properties, and characterization as a zinc metalloenzyme

Brooks, Kathleen P.; Jones, Evan A.; Kim, Byung-Dong; Sander, Eugene G.

doi:10.1016/0003-9861(83)90316-8

Cited by 71 publications

(32 citation statements)

References 22 publications

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“…Several of these enzymes, with different stereoselectivities and substrate specificities, have been used in industrial bioconversion of optically pure D-amino acids with 5-monosubstituted hydantoins as substrates [9,10]. The D-amino acids, as unnatural chiral products, are important intermediates in the synthesis of various compounds, such as the semi-synthetic antibiotic β-lactams, antiviral agents, artificial sweeteners, peptide hormones and pesticides [11][12][13]. The increasing demand of D-amino acids by pharmaceutical and biotechnological companies makes their enzymatic synthesis (via dihydropyrimidinases-hydantoinases) a process which has attracted much attention.…”

Section: Introductionmentioning

confidence: 99%

Metal-triggered changes in the stability and secondary structure of a tetrameric dihydropyrimidinase: A biophysical characterization

Martínez‐Rodríguez

Encinar

Hurtado-Gómez

et al. 2009

Biophysical Chemistry

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This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. A C C E P T E D M A N U S C R I P T ACCEPTED MANUSCRIPT A C C E P T E D M A N U S C R I P T ACCEPTED MANUSCRIPT A C C E P T E D M A N U S C R I P T ACCEPTED MANUSCRIPT 3 ABSTRACTDihydropyrimidinase is involved in the reductive pathway of pyrimidine degradation, catalysing the reversible hydrolysis of the cyclic amide bond (-CO-NH-) of 5,6-dihydrouracil and 5,6-dihydrothymine to the corresponding N-carbamoyl-β-amino acids. This enzyme is an attractive candidate for commercial production of D-amino acids, which are used in the production of semisynthetic β-lactams, antiviral agents, artificial sweeteners, peptide hormones and pesticides. We have obtained the crystal structure of the dihydropyrimidinase from Sinorhizobium meliloti (SmelDhp) in the presence of zinc ions, but we have not been able to obtain good diffracting crystals in its absence. Then, the role of the ion in the structure of the protein, and in its stability, remains to be elucidated. In this work, the stability and the structure of SmelDhp have been studied in the absence and in the presence of zinc. In its absence, the protein acquired a tetrameric functional structure at pH ~6.0, which is stable up to pH ~9.0, as concluded from fluorescence and CD.Chemical-denaturation occurred via a monomeric intermediate with non-native structure. The addition of zinc caused: (i) an increase of the helical structure, and changes in the environment of aromatic residues; and, (ii) a higher thermal stability. However, chemical-denaturation still occurred through a monomeric intermediate. This is the first hydantoinase whose changes in the stability and in the secondary structure upon addition of zinc are described and explained, and one of the few examples where the zinc exclusively alters the secondary helical structure and the environment of some aromatic residues in the protein, leaving unchanged the quaternary structure.

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Section: Introductionmentioning

confidence: 99%

Metal-triggered changes in the stability and secondary structure of a tetrameric dihydropyrimidinase: A biophysical characterization

Martínez‐Rodríguez

Encinar

Hurtado-Gómez

et al. 2009

Biophysical Chemistry

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“…After centrifugation, the supernatant fraction was used for analysis of dihydropyrimidinase and P-ureidopropionase (EC 3.5.1 .6) activities. Dihydropyrimidinase activity was determined by measuring the rate of decrease in absorbance associated with 5-bromo-5,6-dihydrouracil at 225 nm in a cuvette with a 1-0 an light path at 37" (Brooks et al 1983). The standard reaction mixture contained 50 mM-Tris-HC1 buffer, pH 8.2, and 0.17 m~-5-bromo-5,6-dihydrouracil in a total volume of 3-0 ml.…”

Section: Enzyme Assaysmentioning

confidence: 99%

“…Mammalian dihydropyrimidinase was originally studied (Wallach & Grisolia, 1957), purified and characterized in calves (Maguire & Dudley, 1978;Kautz & Schnackerz, 1989), cows (Brooks et al 1983;Lee et al 1986Lee et al ,1987, and rats (Maguire & Dudley, 1978;Kikugawa et al 1994).…”

mentioning

confidence: 99%

“…Dihydropyrimidinase is a tetramer and a Zn-metalloenzyme (Brooks et al 1979(Brooks et al , 1983. The Zn cation can be removed by incubation with the chelators O-phenanthroline, 8-hydroxyquinoline-5-sulphonic acid, or dipicolinic acid, yielding an inactive apoenzyme (Brooks et al 1979(Brooks et al , 1983Lee et al 1986).…”

mentioning

confidence: 99%

“…The Zn cation can be removed by incubation with the chelators O-phenanthroline, 8-hydroxyquinoline-5-sulphonic acid, or dipicolinic acid, yielding an inactive apoenzyme (Brooks et al 1979(Brooks et al , 1983Lee et al 1986). The enzyme activity can be restored by incubating the apoenzyme with Zn (Brooks e l al.…”

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confidence: 99%

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Analysis of cyclic feed intake in rats fed on a zinc-deficient diet and the level of dihydropyrimidinase (EC3.5.2.2)

et al. 1995

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The body weight and feed intake of rats fed on a Zn-deficient diet for 28 d were reduced compared with those of control rats. The feed intakes of the Zn-deficient and control groups during the period were 10.2 (SE 0.3) and 15-7 (SE 0.2) g/d respectively. Cyclic variations in feed intake and body-weight changes were found in analysis not only of all the data for five rats but also that in each individual rat. Cosinor analysis revealed that the cyclical period of both the feed intake and body-weight change in the Zn-deficient rats was 3.5 (SE 0.1) d. The mesor and amplitude value of the feed intake in the Zn-deficient rats was 10.1 (SE 0.4) g/d and 3 5 (SE 0.5) g/d respectively, and that of body-weight change was 1.4 (SE 0.1) g/d and 7.9 (SE 1.3) g/d respectively. Among pyrimidine-catabolizing enzymes, dihydropyrimidinase (EC 3.5.2.2) activity showed significant retardation in the Zn-deficient rat liver with decrease of the enzyme protein.The ratio of apo-form to holo-form dihydropyrimidinase in the liver was not alfected by the Zndeficient diet.Zinc deficiency : Cosinor analysis : Dihydropyrimidinase

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Stereoselective metabolism of dexrazoxane (ICRF-187) and levrazoxane (ICRF-186)

Hasinoff

Aoyama

1999

Chirality

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Bovine liver dihydropyrimidine amidohydrolase: Purification, properties, and characterization as a zinc metalloenzyme

Cited by 71 publications

References 22 publications

Metal-triggered changes in the stability and secondary structure of a tetrameric dihydropyrimidinase: A biophysical characterization

Metal-triggered changes in the stability and secondary structure of a tetrameric dihydropyrimidinase: A biophysical characterization

Analysis of cyclic feed intake in rats fed on a zinc-deficient diet and the level of dihydropyrimidinase (EC3.5.2.2)

Stereoselective metabolism of dexrazoxane (ICRF-187) and levrazoxane (ICRF-186)

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