Proteolytic cleavage of the hyperthermophilic topoisomerase I from Thermotoga maritima does not impair its enzymatic properties - Archive ouverte HAL Access content directly
Journal Articles BBA - Biochimica et Biophysica Acta Year : 2004

Proteolytic cleavage of the hyperthermophilic topoisomerase I from Thermotoga maritima does not impair its enzymatic properties

Abstract

Using limited proteolysis, we show that the hyperthermophilic topoisomerase I from Thermotoga maritima exhibits a unique hot spot susceptible to proteolytic attack with a variety of proteases. The remaining of the protein is resistant to further proteolysis, which suggests a compact folding of the thermophilic topoisomerase, when compared to its mesophilic Escherichia coli homologue. We further show that a truncated version of the T. maritima enzyme, lacking the last C-terminal 93 amino acids is more susceptible to proteolysis, which suggests that the C-terminal region of the topoisomerase may be important to maintain the compact folding of the enzyme. The hot spot of cleavage is located around amino acids 326-330 and probably corresponds to an exposed loop of the protein, near the active site tyrosine in charge of DNA cleavage and religation. Location of this protease sensitive region in the vicinity of bound DNA is consistent with the partial protection observed in the presence of different DNA substrates. Unexpectedly, although proteolysis splits the enzyme in two halves, each containing part of the motifs involved in catalysis, trypsin-digested topoisomerase I retains full DNA binding, cleavage, and relaxation activities, full thermostability and also the same hydrodynamic and spectral properties as undigested samples. This supports the idea that the two fragments which are generated by proteolysis remain correctly folded and tightly associated after proteolytic cleavage.

Dates and versions

hal-04135464 , version 1 (20-06-2023)

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Raynald Cossard, Thierry Viard, Valérie Lamour, Michel Duguet, Claire Bouthier de La Tour. Proteolytic cleavage of the hyperthermophilic topoisomerase I from Thermotoga maritima does not impair its enzymatic properties. BBA - Biochimica et Biophysica Acta, 2004, 1700 (2), pp.161-170. ⟨10.1016/j.bbapap.2004.04.009⟩. ⟨hal-04135464⟩
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