Protein Engineering, Vol. 14, No. 10, 807-813,
October 2001
© 2001 Oxford University Press
Cytidine deaminase from two extremophilic bacteria: cloning, expression and comparison of their structural stability
1 Department of Compared Morphological and Biochemical Sciences, 2 Department of Veterinary Sciences, 3 Department of MCA Biology, University of Camerino, Matelica, Italy and 4 Institute of Molecular Biology, University of Copenhagen, Copenhagen, Denmark
We cloned, purified and characterized two extremophilic cytidine deaminases: CDABcald and CDABpsy, isolated from Bacillus caldolyticus (growth at 72°C) and Bacillus psychrophilus (growth at 10°C), respectively. We compared their thermostability also with the mesophilic counterpart, CDABsubt, isolated from Bacillus subtilis (growth at 37°C). The DNA fragments encoding CDABcald and CDABpsy were sequenced and the deduced amino acid sequences showed 70% identity. High sequence similarity was also found with the mesophilic CDABsubt. Both enzymes were found to be homotetramers of approximately 58 kDa. CDABcald was found to be highly thermostable, as expected, up to 65°C, whereas CDABpsy showed higher specific activity at lower temperatures and was considerably less thermostable than CDABcald. After partial denaturation at 72°C for 30 min, followed by renaturation on ice, CDABcald recovered 100% of its enzymatic activity, whereas CDABpsy as well as CDABsubt were irreversibly inactivated. Circular dichroism (CD) spectra of CDABcald and CDABpsy at temperatures ranging from 10 to 95°C showed a markedly different thermostability of their secondary structures: at 10 and 25°C the CD spectra were indistinguishable, suggesting a similar overall structure, but as temperature increases up to 50–70°C, the 
-helices of CDABpsy unfolded almost completely, whereas its ß-structure and the aromatic amino acids core remained pretty stable. No significant differences were seen in the secondary structures of CDABcald with increase in temperature.