Incomplete Factorial Search for Conditions Leading to High Quality Crystals of Escherichia coli Cytidine Deaminase Complexed to a Transition State Analog Inhibitor

Betts, Laurie; Frick, Lloyd; Wolfenden, Richard; Carter, C W

doi:10.1016/s0021-9258(18)83491-3

Cited by 20 publications

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References 23 publications

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“…To uncover the structural basis of these remarkable powers of binding discrimination and to assist the solution of the crystal structure of bacterial cytidine deaminase, it would be desirable to learn the amino acid sequence of bacterial cytidine deaminase. The amino acid sequence and three-dimensional structure of mouse adenosine deaminase have been reported recently (Chang et al, 1991;Wilson et al, 1991), and crystals of bacterial cytidine deaminase have been prepared in the presence and absence of the transition-state analogue inhibitor 5-fluoropyrimidin-2-one ribonucleoside (Betts et al, 1989). This paper describes the cloning of the cdd gene that encodes cytidine deaminase in Escherichia coli K-12 and the determination of its DNA sequence.…”

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Cloning and nucleotide sequence of the Escherichia coli cytidine deaminase (ccd) gene

Yang¹,

Carlow²,

Wolfenden

et al. 1992

Biochemistry

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The structural gene that encodes cytidine deaminase (cdd) in Escherichia coli was cloned from Kohara phage lambda 365 (7F1), and its nucleotide sequence was determined. Plasmids harboring the gene complemented chromosomal cdd mutations, enhanced cytidine deaminase activity in cell extracts, and directed the synthesis of a protein identical in mass and N-terminal amino acid sequence with cytidine deaminase purified from wild-type bacteria. Metal analysis of the purified, plasmid-encoded deaminase indicated a single atom of tightly bound zinc per subunit. Earlier work has shown that bacterial cytidine deaminase and mammalian adenosine deaminase are remarkably alike in their mechanisms of action, in their free energies of interaction with analogue inhibitors resembling tetrahedral intermediates in nucleophilic substitution, and in their ability to discriminate between analogue inhibitors differing by a single hydroxyl group. In contrast to these shared catalytic similarities, the deduced amino acid sequence of E. coli cytidine deaminase (monomer MW 31,540) differs markedly from the mammalian adenosine deaminase sequence suggesting major differences in their tertiary structures. Nevertheless, cytidine deaminase and mammalian plus bacterial adenosine deaminases share a single region (TVHA) of sequence identity that is tentatively identified as part of the cytidine deaminase active site.

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confidence: 99%

Cloning and nucleotide sequence of the Escherichia coli cytidine deaminase (ccd) gene

Yang¹,

Carlow²,

Wolfenden

et al. 1992

Biochemistry

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Crystallization, Bulk, Macromolecules

Rosa

McPherson

1999

Encyclopedia of Bioprocess Technology

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Introduction The Uniqueness of Macromolecular Crystals Methods for Growth of Protein Crystals Methods for Promoting Supersaturation The Crystallization of Proteins by Variation of pH or Temperature Bibliography

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Crystallization of yeast orotidine 5′‐monophosphate decarboxylase complexed with 1‐(5′‐phospho‐β‐D‐ribofuranosyl) barbituric acid

Bell¹,

Jones²,

Carter³

1991

Proteins

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Using an incomplete factorial experimental design, we have identified conditions for crystallization of yeast orotidine 5'-monophosphate decarboxylase (ODCase) in an unliganded state and complexed separately to two inhibitors: 6-azauridine 5'-monophosphate (aza-UMP) and 1-(5'-phospho-beta-D-ribofuranosyl) barbituric acid (BMP). Crystals of X-ray diffraction quality have been obtained of yeast ODCase complexed with BMP, a putative transition state analog inhibitor (Ki = 8.8 x 10(-12) M). ODCase:BMP complex crystals with a hexagonal rod habit were grown from a solution initially containing 12 mg/ml ODCase (205 microM dimer) plus 450 microM BMP by microdialysis at 4 degrees C against a mother liquor which consisted of 0.1 M Na-PIPES-acetate (pH 6.4), 37.5 microM BMP, 5 mM mercaptoethanol, 1% polyethylene glycol 400, and 2.3 M ammonium sulfate. Crystals were analyzed using precession photography and were assigned to trigonal space group R32 with unit cell dimensions a = b = 115 A, c = 385 A. The crystal density is 1.245 g/cm3 indicating the presence of two ODCase: BMP complex dimers (118 kDa each) per asymmetric unit with a packing density of 2.08 A3/Da and 41% solvent content. The morphological habit of crystals of the ODCase:BMP complex changed when the initial ammonium sulfate concentration was increased in 0.05 M steps from 2.3 to 2.45 M. All of these crystals diffracted to at least 3.0 A resolution over a period of several weeks at room temperature and are isomorphous.

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Incomplete Factorial Search for Conditions Leading to High Quality Crystals of Escherichia coli Cytidine Deaminase Complexed to a Transition State Analog Inhibitor

Cited by 20 publications

References 23 publications

Cloning and nucleotide sequence of the Escherichia coli cytidine deaminase (ccd) gene

Cloning and nucleotide sequence of the Escherichia coli cytidine deaminase (ccd) gene

Crystallization, Bulk, Macromolecules

Crystallization of yeast orotidine 5′‐monophosphate decarboxylase complexed with 1‐(5′‐phospho‐β‐D‐ribofuranosyl) barbituric acid

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