Cofactor binding modulates the conformational stabilities and unfolding patterns of NAD(+)-dependent DNA ligases from Escherichia coli and Thermus scotoductus.
DNA ligases are important enzymes required for cellular processes such as DNA replication, recombination, and repair. NAD(+)-dependent DNA ligases are essentially restricted to eubacteria, thus constituting an attractive target in the development of novel antibiotics. Although such a project might involve the systematic testing of a vast number of chemical compounds, it can essentially gain from the preliminary deciphering of the conformational stability and structural perturbations associated with the formation of the catalytically active adenylated enzyme. We have, therefore, investigated the adenylation-induced conformational changes in the mesophilic Escherichia coli and thermophilic Thermus scotoductus NAD(+)-DNA ligases, and the resistance of these enzymes to thermal and chemical (guanidine hydrochloride) denaturation. Our results clearly demonstrate that anchoring of the cofactor induces a conformational rearrangement within the active site of both mesophilic and thermophilic enzymes accompanied by their partial compaction. Furthermore, the adenylation of enzymes increases their resistance to thermal and chemical denaturation, establishing a thermodynamic link between cofactor binding and conformational stability enhancement. Finally, guanidine hydrochloride-induced unfolding of NAD(+)-dependent DNA ligases is shown to be a complex process that involves accumulation of at least two equilibrium intermediates, the molten globule and its precursor.
Georlette
Daphné
D
Laboratory of Biochemistry, Institute of Chemistry B6, University of Liège, B-4000 Liège, Belgium.
Blaise
Vinciane
V
Dohmen
Christophe
C
Bouillenne
Fabrice
F
Damien
Benjamin
B
Depiereux
Eric
E
Gerday
Charles
C
Uversky
Vladimir N
VN
Feller
Georges
G
D000900:Anti-Bacterial Agents / Q000494:pharmacology; D002151:Calorimetry; D011088:DNA Ligases / Q000737:chemistry* / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004926:Escherichia coli / Q000201:enzymology*; D019791:Guanidine / Q000737:chemistry / Q000494:pharmacology; D009243:NAD / Q000737:chemistry*; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D017510:Protein Folding; D013050:Spectrometry, Fluorescence; D013696:Temperature; D013816:Thermodynamics; D013824:Thermus / Q000201:enzymology*
D000900:Anti-Bacterial Agents; D009243:NAD; D011088:DNA Ligases; C120640:DNA ligase (NAD); D019791:Guanidine