Thomas Lautier1, Philippe Urban1, Jacqueline Loeper2, Laetitia Jezequel3, Denis Pompon1, Gilles Truan4. 1. Université de Toulouse, INSA, UPS, INP, LISBP, 135 Avenue de Rangueil, F-31077 Toulouse, France; INRA, UMR792 Ingénierie des Systèmes Biologiques et des Procédés, F-31400 Toulouse, France; CNRS, UMR5504, 135 Avenue de Rangueil, F-31400 Toulouse, France. 2. Université Pierre-et-Marie-Curie, 4 Place Jussieu, 75005 Paris, France; INSERM U773, Faculté de Médecine Bichat-Beaujon, 75018 Paris, France. 3. Solvay, Product Compliance, 52 rue de la Haie-Coq, 93306 Aubervilliers, France. 4. Université de Toulouse, INSA, UPS, INP, LISBP, 135 Avenue de Rangueil, F-31077 Toulouse, France; INRA, UMR792 Ingénierie des Systèmes Biologiques et des Procédés, F-31400 Toulouse, France; CNRS, UMR5504, 135 Avenue de Rangueil, F-31400 Toulouse, France. Electronic address: gilles.truan@insa-toulouse.fr.
Abstract
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-trifluoromethylcoumarin. 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.
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-trifluoromethylcoumarin. 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.
Authors: Dominique A Ramirez; Keagan P Collins; Allister E Aradi; Katherine A Conger; Daniel L Gustafson Journal: Drug Metab Dispos Date: 2018-12-19 Impact factor: 3.922
Authors: Ran Zuo; Yi Zhang; Chao Jiang; John C Hackett; Rosemary Loria; Steven D Bruner; Yousong Ding Journal: Sci Rep Date: 2017-04-12 Impact factor: 4.379