Cytidine deaminase from two extremophilic bacteria: cloning, expression and comparison of their structural stability

We cloned, purified and characterized two extremophilic cytidinedeaminases: CDA_Bcald and CDA_Bpsy, isolated from Bacillus caldolyticus(growth at 72°C) and Bacillus psychrophilus (growth at 10°C),respectively. We compared their thermostability also with themesophilic counterpart, CDA_Bsubt, isolated from Bacillus subtilis(growth at 37°C). The DNA fragments encoding CDA_Bcald andCDA_Bpsy were sequenced and the deduced amino acid sequencesshowed 70% identity. High sequence similarity was also foundwith the mesophilic CDA_Bsubt. Both enzymes were found to behomotetramers of approximately 58 kDa. CDA_Bcald was found tobe highly thermostable, as expected, up to 65°C, whereasCDA_Bpsy showed higher specific activity at lower temperaturesand was considerably less thermostable than CDA_Bcald. Afterpartial denaturation at 72°C for 30 min, followed by renaturationon ice, CDA_Bcald recovered 100% of its enzymatic activity, whereasCDA_Bpsy as well as CDA_Bsubt were irreversibly inactivated. Circulardichroism (CD) spectra of CDA_Bcald and CDA_Bpsy at temperaturesranging from 10 to 95°C showed a markedly different thermostabilityof their secondary structures: at 10 and 25°C the CD spectrawere indistinguishable, suggesting a similar overall structure,but as temperature increases up to 50–70°C, the -helicesof CDA_Bpsy unfolded almost completely, whereas its ß-structureand the aromatic amino acids core remained pretty stable. Nosignificant differences were seen in the secondary structuresof CDA_Bcald with increase in temperature.