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

The complementary DNA (cDNA) coding for Arabidopsis thaliana cytidine deami nase 1 (AT-CDA1) was obtained from the amplified A. thaliana cDNA expressio n library, provided by R. W. Davis (Stanford University, CA). AT-CDA1 cDNA was subcloned into the expression vector pTrc99-A and the protein, expresse d in Escherichia coli following induction with isopropyl 1-thio-beta-D-gala ctopyranoside, showed high cytidine deaminase activity. The nucleotide sequ ence 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 s equence of AT-CDA1 showed no transit peptide for targeting to the chloropla st or mitochondria indicating that this form of cytidine deaminase is proba bly expressed in the cytosol. The recombinant AT-CDA1 was purified to homog eneity 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 s pecific 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 CDA s, that the enzyme is a dimer composed of two identical subunits. Inductive ly coupled plasma-optical emission spectroscopy analysis indicated that the enzyme contains 1 mol of zinc atom per mole of subunit. The kinetic proper ties of AT-CDA1 both toward the natural substrates and with analogs indicat ed that the catalytic mechanism of the plant enzyme is probably very simila r to that of the human the E. coli enzymes. (C) 1999 Academic Press.