Cloning, Expression, and Purification of Cytidine Deaminase from Arabidopsis thaliana Silvia Vincenzetti,* Alessandra Cambi,* Jan Neuhard,† Kirk Schnorr,† ,1 Mauro Grelloni,* and Alberto Vita* ,2 *Dipartimento di Scienze Morfologiche e Biochimiche Comparate, Universita ` di Camerino, Camerino (MC), Italy; and †Centre for Enzyme Research, University of Copenhagen, Denmark Received May 8, 1998, and in revised form July 15, 1998 The complementary DNA (cDNA) coding for Arabi- dopsis thaliana cytidine deaminase 1 (AT-CDA1) was obtained from the amplified A. thaliana cDNA expres- sion library, provided by R. W. Davis (Stanford Univer- sity, CA). AT-CDA1 cDNA was subcloned into the ex- pression vector pTrc99-A and the protein, expressed in Escherichia coli following induction with isopropyl 1-thio--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 chloro- plast or mitochondria indicating that this form of cy- tidine deaminase is probably expressed in the cytosol. The recombinant AT-CDA1 was purified to homogene- ity 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 spe- cific activity of 74 U/mg. The molecular mass of AT- CDA1 estimated by gel filtration was 63 kDa, indicat- ing, in contrast to the other eukaryotic CDAs, that the enzyme is a dimer composed of two identical subunits. Inductively coupled plasma– optical emission spectro- scopy 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. © 1999 Academic Press Cytidine deaminase (CDA 3 ; EC 3.5.4.5) catalyzes the hydrolytic deamination of cytidine and deoxycytidine to uridine and deoxyuridine, respectively. Further- more, the enzyme is capable of deaminating a number of cytotoxic cytosine nucleoside analogs such as arabi- nosyl cytosine, 5-azadeoxycytidine, and many others, causing their pharmacological inactivation (1,2). Based on deduced amino acid sequences obtained from the databanks, the size of the CDA subunits from different organisms is either approximately 30 kDa as for Esch- erichia coli (3) and Haemophilus influenzae (4) or half of that, i.e., approximately 15 kDa as for Mycoplasma pneumoniae (5), Bacillus subtilis (6), chicken (7), and human (8). Despite the differences in subunit size the molecular mass of the native enzymes from E. coli, B. subtilis, chicken, and human were found to be 60 – 64 kDa. Thus, E. coli CDA is a homodimer (2  32 kDa), whereas the enzymes from B. subtilis, chicken, and human are homotetramers (4  15 kDa). CDAs from E. coli (9), chicken (7), sheep (10), mouse (11), and various human tissues (12–14) were studied in some detail, finding quite similar kinetic properties. The crystal structure of the dimeric E. coli enzyme has been determined (15). It showed that the enzyme con- tained one tightly bound zinc per subunit in the active site and that the zinc atom was coordinated by one histidine and two cysteine residues. The structure fur- ther indicated that each monomer was composed of a small N-terminal -helical domain and two core do- mains and that the two core domains showed nearly 1 Present address: Screening Biotechnology, Novo Nordisk A/S, Bagsvaerd, Denmark. 2 To whom correspondence should be addressed at Dipartimento di Scienze Morfologiche e Biochimiche Comparate, Universita ` di Cam- erino, via Camerini 3, 62032 Camerino (MC), Italy. Fax: (+39)/(737)/ 403217; E-mail: vita@cambio.unicam.it. 3 Abbreviations used: AT-CDA1, Arabidopsis thaliana cytidine deaminase 1; CV6, 6-[3-(5-cytidyl) acryloylamino]hexanoic acid; cDNA, complementary DNA; DTT, dithiothreitol; ICP–OES, induc- tively coupled plasma– optical emission spectroscopy; IPTG, isopro- pyl 1-thio--D-galactopyranoside; PAGE, polyacrylamide gel electro- phoresis; PCR, polymerase chain reaction; SDS, sodium dodecyl sulfate. Protein Expression and Purification 15, 8 –15 (1999) Article ID prep.1998.0959, available online at http://www.idealibrary.com on 8 1046-5928/99 $30.00 Copyright © 1999 by Academic Press All rights of reproduction in any form reserved.