We cloned, purified and characterized two extremophilic cytidine deaminases: CDA(Bcald) and CDA(Bpsy), isolated from Bacillus caldolyticus (growth at 72 degrees C) and Bacillus psychrophilus (growth at 10 degrees C), respectively. We compared their thermostability also with the mesophilic counterpart, CDA(Bsubt), isolated from Bacillus subtilis (growth at 37 degrees C). The DNA fragments encoding CDA(Bcald) and CDA(Bpsy) were sequenced and the deduced amino acid sequences showed 70% identity. High sequence similarity was also found with the mesophilic CDA(Bsubt). Both enzymes were found to be homotetramers of approximately 58 kDa. CDA(Bcald) was found to be highly thermostable, as expected, up to 65 degrees C, whereas CDA(Bpsy) showed higher specific activity at lower temperatures and was considerably less thermostable than CDA(Bcald). After partial denaturation at 72 degrees C for 30 min, followed by renaturation on ice, CDA(Bcald) recovered 100% of its enzymatic activity, whereas CDA(Bpsy) as well as CDA(Bsubt) were irreversibly inactivated. Circular dichroism (CD) spectra of CDA(Bcald) and CDA(Bpsy) at temperatures ranging from 10 to 95 degrees C showed a markedly different thermostability of their secondary structures: at 10 and 25 degrees C the CD spectra were indistinguishable, suggesting a similar overall structure, but as temperature increases up to 50-70 degrees C, the alpha-helices of CDA(Bpsy) unfolded almost completely, whereas its beta-structure and the aromatic amino acids core remained pretty stable. No significant differences were seen in the secondary structures of CDA(Bcald) with increase in temperature. Study holds ProTherm entries: 12768, 12769, 12770 Extra Details: circular dichroism; cytidine deaminase; extremophiles; thermostability
Submitter: Connie Wang
Submission Date: April 24, 2018, 8:44 p.m.
|Number of data points||3|
|Proteins||Cytidine deaminase ; Cytidine deaminase ; Cytidine deaminase ; Cytidine deaminase|
|Assays/Quantities/Protocols||Experimental Assay: dG_H2O|
|Libraries||Mutations for sequence MEIEQLIVEAKKARELAYVPYSKFPVGAALLTKGGSVYRGCNIENAAYSVCNCAERTALFKAYSEGEKEFTALAVIADTPRPVPPCGACRQVIAELCHGDMKVILANLKGDVKVMTVSELLPEAFSAEDLHA ; Mutations for sequence MNRQELITEALKARDMAYAPYSKFQVGAALLTKDGKVYRGCNIENAAYSMCNCAERTALFKAVSEGDTEFQMLAVAADTPGPVSPCGACRQVISELCTKDVIVVLTNLQGQIKEMTVEELLPGAFSSEDLHDERKL ; Mutations for sequence MDVEKLIAESKKAREQAYVPYSKFPVGAALLAEDGTIYHGCNIENSAYSMTNCAERTAFFKAVSDGVRSFKALAVVADTEGPVSPCGACRQVIAEFCNGSMPVYLTNLKGDIEETTVAKLLPGAFSKEDLSYAAEQ|