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Article Dans Une Revue Biochimica et Biophysica Acta (BBA) - General Subjects Année : 2016

Ordered chimerogenesis applied to CYP2B P450 enzymes

Résumé

Background: Structural studies on CYP2B enzymes identified some of the features that are related to their high plasticity. The aim of this work was to understand further the possible relationships between combinations of structural elements and functions by linking shift in substrate specificity with sequence element swaps between CYP2B6 and CYP2B11. Methods: A series of 15 chimeras in which a small CYP2B6 sequence segment was swapped with its equivalent in CYP2B11 were constructed. All chimeras produced were thus mostly of CYP2B11 sequence. Time course studies were carried out with two typical CYP2B substrates, cyclophosphamide and 7-ethoxy-4-trifiuoromethylcoumarin. Steady-state kinetic parameters were determined for all chimeras expressed in yeast. Results: Most of the chimeras exhibit a high affinity for cyclophosphamide, as CYP2B11 does. A few exhibit an affinity similar to that of CYP2B6 without altered behavior toward the other substrate assayed. The swapped elements that control this specificity shift are discussed in terms of F'/G' cassette role and substrate access channels. Conclusions: Some sequence segments control precisely the shift in affinity for cyclophosphamide between CYP2B6, which has a typical low affinity, and CYP2B11 which has a typical high affinity. General significance: The result provides a new basis for determining the structural elements that control functions in complex enzymes.
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Dates et versions

hal-01886421 , version 1 (02-10-2018)

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Thomas Lautier, Philippe Urban, Jacqueline Loeper, Laetitia Jezequel, Denis Pompon, et al.. Ordered chimerogenesis applied to CYP2B P450 enzymes. Biochimica et Biophysica Acta (BBA) - General Subjects, 2016, 1860 (7), pp.1395-1403. ⟨10.1016/j.bbagen.2016.03.028⟩. ⟨hal-01886421⟩
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