A DNA ligase from the psychrophile Pseudoalteromonas haloplanktis gives insights into the adaptation of proteins to low temperatures
2000
Georlette, D. | Jónsson, Z. O. | Van Petegem, F. | Chessa, J.âP. | Van Beeumen, J. | Hübscher, U. | Gerday, C.
The cloning, overexpression and characterization of a coldâadapted DNA ligase from the Antarctic sea water bacterium Pseudoalteromonas haloplanktis are described. Protein sequence analysis revealed that the coldâadapted Ph DNA ligase shows a significant level of sequence similarity to other NAD+âdependent DNA ligases and contains several previously described sequence motifs. Also, a decreased level of arginine and proline residues in Ph DNA ligase could be involved in the coldâadaptation strategy. Moreover, 3D modelling of the Nâterminal domain of Ph DNA ligase clearly indicates that this domain is destabilized compared with its thermophilic homologue. The recombinant Ph DNA ligase was overexpressed in Escherichia coli and purified to homogeneity. Mass spectroscopy experiments indicated that the purified enzyme is mainly in an adenylated form with a molecular mass of 74â593âDa. Ph DNA ligase shows similar overall catalytic properties to other NAD+âdependent DNA ligases but is a coldâadapted enzyme as its catalytic efficiency (kcat/Km) at low and moderate temperatures is higher than that of its mesophilic counterpart E.âcoli DNA ligase. A kinetic comparison of three enzymes adapted to different temperatures (P.âhaloplanktis, E.âcoli and Thermus scotoductus DNA ligases) indicated that an increased kcat is the most important adaptive parameter for enzymatic activity at low temperatures, whereas a decreased Km for the nicked DNA substrate seems to allow T.âscotoductus DNA ligase to work efficiently at high temperatures. Besides being useful for investigation of the adaptation of enzymes to extreme temperatures, P.âhaloplanktis DNA ligase, which is very efficient at low temperatures, offers a novel tool for biotechnology.
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