Publications

• Chromosomal location, cloning and nucleotide sequence of the Bacillus subtilis cdd gene encoding cytidine/deoxycytidine deaminase.

  Song B.-H., Neuhard J.

  The Bacillus subtilis cdd gene encoding cytidine/2'-deoxycytidine deaminase has been located by transduction at approximately 225 degrees on the chromosome, and the gene order trpC-lys-cdd-aroD was established. The gene was isolated from a library of B. subtilis DNA cloned in lambda D69 by complem ... >> More

  The Bacillus subtilis cdd gene encoding cytidine/2'-deoxycytidine deaminase has been located by transduction at approximately 225 degrees on the chromosome, and the gene order trpC-lys-cdd-aroD was established. The gene was isolated from a library of B. subtilis DNA cloned in lambda D69 by complementation of an Escherichia coli cdd mutation. Minicell experiments revealed a molecular mass of 14,000 dalton for the cytidine deaminase subunit encoded by the cloned DNA fragment. The molecular weight of the native enzyme was determined to be 58,000, suggesting that it consists of four identical subunits. The nucleotide sequence of 1170 bp, including the cdd gene, was determined. An open reading frame encoding a polypeptide with a calculated molecular mass of 14,800 dalton was deduced to be the coding region for cdd. The deduced amino acid composition of the 136-amino acid-long subunit shows that it contains six cysteine residues. A computer search in the GenBank DNA sequence library revealed that the 476 bp HindIII fragment containing the putative promoter region and the first ten codons of cdd is identical to the P43 promoter-containing fragment previously isolated by Wang and Doi (1984). They showed that the fragment contained overlapping promoters transcribed by B. subtilis sigma 43 and sigma 37 RNA polymerase holoenzymes during growth and stationary phase. << Less

  Mol. Gen. Genet. 216:462-468(1989) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• Cloning, expression, and purification of cytidine deaminase from Arabidopsis thaliana.

  Vincenzetti S., Cambi A., Neuhard J., Schnorr K., Grelloni M., Vita A.

  The complementary DNA (cDNA) coding for Arabidopsis thaliana cytidine deaminase 1 (AT-CDA1) was obtained from the amplified A. thaliana cDNA expression library, provided by R. W. Davis (Stanford University, CA). AT-CDA1 cDNA was subcloned into the expression vector pTrc99-A and the protein, expres ... >> More

  The complementary DNA (cDNA) coding for Arabidopsis thaliana cytidine deaminase 1 (AT-CDA1) was obtained from the amplified A. thaliana cDNA expression library, provided by R. W. Davis (Stanford University, CA). AT-CDA1 cDNA was subcloned into the expression vector pTrc99-A and the protein, expressed in Escherichia coli following induction with isopropyl 1-thio-beta-d-galactopyranoside, showed high cytidine deaminase activity. The nucleotide sequence showed a 903-bp open reading frame encoding a polypeptide of 301 amino acids with a calculated molecular mass of 32,582. The deduced amino acid sequence of AT-CDA1 showed no transit peptide for targeting to the chloroplast or mitochondria indicating that this form of cytidine deaminase is probably expressed in the cytosol. The recombinant AT-CDA1 was purified to homogeneity by a heat treatment followed by an ion-exchange chromatography. The final enzyme preparation was >98% pure as judged by SDS-PAGE and showed a specific activity of 74 U/mg. The molecular mass of AT-CDA1 estimated by gel filtration was 63 kDa, indicating, in contrast to the other eukaryotic CDAs, that the enzyme is a dimer composed of two identical subunits. Inductively coupled plasma-optical emission spectroscopy analysis indicated that the enzyme contains 1 mol of zinc atom per mole of subunit. The kinetic properties of AT-CDA1 both toward the natural substrates and with analogs indicated that the catalytic mechanism of the plant enzyme is probably very similar to that of the human the E. coli enzymes. << Less

  Protein Expr. Purif. 15:8-15(1999) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• Human cytidine deaminase: purification of enzyme, cloning, and expression of its complementary DNA.

  Laliberte J., Momparler R.L.

  Cytidine (CR) deaminase was purified 47,000-fold to homogeneity from human placenta. The molecular mass of CR deaminase was estimated to be 48.7 kDa by gel filtration and 16.1 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis, suggesting that it contains three or four identical subu ... >> More

  Cytidine (CR) deaminase was purified 47,000-fold to homogeneity from human placenta. The molecular mass of CR deaminase was estimated to be 48.7 kDa by gel filtration and 16.1 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis, suggesting that it contains three or four identical subunits. We determined the amino acid sequence of several peptide fragments and designed 5'-primers to amplify, by the polymerase chain reaction, a specific 364-base pair DNA fragment using human liver complementary DNA (cDNA) as the template. This DNA fragment, which contains the codons of one peptide, was used as a probe to screen a cDNA library from human liver. We isolated and sequenced a cDNA clone of 910 base pairs that contained a 5' nontranslated region, a 438-base pair coding region, and a 3' nontranslated region with a polyadenylated tail. The translated region of the clone contained a deduced sequence of 146 amino acids, with a predicted molecular mass of 16.2 kDa and the sequences of our peptides. The cDNA was ligated in pGEX vector and expressed in Escherichia coli. The expressed protein had a high CR deaminase activity and molecular mass of 16.3 kDa. These data demonstrate clearly that the open reading frame of our cDNA clone codes for a functional human CR deaminase. Polymerase chain reaction amplifications of gene-specific DNA fragments from human/rodent hybrid cells indicate the localization of CR deaminase gene to human chromosome 1. The cDNA for CR deaminase will be a useful molecular probe to investigate the importance of this enzyme in chemotherapy. << Less

  Cancer Res. 54:5401-5407(1994) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• The structure of the cytidine deaminase-product complex provides evidence for efficient proton transfer and ground-state destabilization.

  Xiang S., Short S.A., Wolfenden R., Carter C.W. Jr.

  Crystal structures of the cytidine deaminase-uridine product complex prepared either by cocrystallizing enzyme with uridine or by diffusing cytidine into ligand-free crystals show that the product binds as a 4-ketopyrimidine. They reveal four additional features of the catalytic process. (1) A wat ... >> More

  Crystal structures of the cytidine deaminase-uridine product complex prepared either by cocrystallizing enzyme with uridine or by diffusing cytidine into ligand-free crystals show that the product binds as a 4-ketopyrimidine. They reveal four additional features of the catalytic process. (1) A water molecule bound to a site previously observed to bind the incoming 4-NH2 group represents the site for the leaving ammonia molecule. The conserved Pro 128 accommodates both moieties by orienting the carbonyl group of the previous residue. (2) The Glu 104 carboxylate group rotates from its hydrogen bond to the O4 hydroxyl group in transition-state analog complexes, forming a new hydrogen bond to the leaving group moiety. Thus, after stabilizing the hydroxyl group in the transition state, Glu 104 transfers a proton from that group to the leaving amino group, promoting enol-to-keto isomerization of the product. (3) Difference Fourier comparisons with transition-state complexes indicate that the pyrimidine ring rotates toward the zinc by approximately 10 degrees. The active site thus "pulls" the ring and 4-NH2 group in opposite directions during catalysis. To preserve coplanarity of the 4-keto group with the pyrimidine ring, the N1-C1' glycosidic bond bends by approximately 19 degrees out of the ring plane. This distortion may "spring-load" the product complex and promote dissociation. Failure to recognize a similar distortion could explain an earlier crystallographic interpretation of the adenosine deaminase-inosine complex [Wilson, D. K., & Quiocho, F. A. (1994) Nat. Struct. Biol. 1, 691-694]. (4) The Zn-Sgamma132 bond, which lengthens in transition-state complexes, shortens as the O4 atom returns to a state of lower negative charge in the planar product, consistent with our previous proposal that this bond buffers the zinc bond valence, compensating buildup of negative charge on the oxygen nucleophile during catalysis. << Less

  Biochemistry 36:4768-4774(1997) [PubMed] [EuropePMC]