Active Site Crowding of Cytochrome P450 3A4 as a Strategy To Alter Its Selectivity

Abstract

Substrate-promiscuous enzymes are a promising starting point for the development of versatile biocatalysts. In this study, human cytochrome P450 3A4, known for its ability to metabolise hundreds of drugs, was engineered to alter its regio- and stereoselectivity. Rational mutagenesis was used to introduce steric hindrance in a specific manner in the large active site of P450 3A4 and to favour oxidation at a more sterically accessible position on the substrate. Hydroxylation of a synthetic precursor of (R)-lisofylline, a compound under investigation for its anti-inflammatory properties, was chosen as a first proof-of-principle application of our protein engineering strategy. In a second example, increasing active site crowding led to an incremental shift in the selectivity of oxidation from an internal double bond to a terminal phenyl group in a derivative of theobromine. The same correlation between crowding and selectivity was found in a final case focused on the hydroxylation of the steroid sex hormone progesterone.

Second‐Coordination Sphere Effects on Selectivity and Specificity of Heme and Nonheme Iron Enzymes

“…For instance, with theobromine as a substrate, the wildtype structure gives 19 % C 5 ‐hydroxylation and 81 % C 4 ‐hydroxylation as products. However, studies with the Ala370Ile or Ala370Leu variants gave a regioselectivity reversal and produced predominantly C 5 ‐hydroxylation instead . Similarly, with triple or quintuple mutants of P450 3A4, the activation of progesterone gave dominant 2β‐hydroxylation rather than 6β‐hydroxylation and, hence, affected substrate positioning and activation …”

Selective Oxidations of Cyperenoic Acid by Slightly Reshaping the Binding Pocket of Cytochrome P450 BM3

“…Notably, a major reason for the substrate promiscuity of P450s is the plasticity of the large and nonpolar substrate binding pocket . Adjustment of topological structure can substantially influence the selectivity of P450‐catalyzed hydroxylation . Hence, the construction of the mutant library was guided by two basic hypotheses.…”

“…[26] Adjustment of topological structure can substantially influence the selectivity of P450catalyzed hydroxylation. [27] Hence, the construction of the mutant library was guided by two basic hypotheses.F irst, we presumed that enzymea ctivity was going to be best maintained by replacing nonpolars ide chains with similar nonpolar amino acids. Second, mutationsthat simply changed steric hindrance could result in different selectivity.T hus, selected amino acids were replaced by hydrophobic amino acids that have smaller or biggerv an der Waals volumes.…”

Computational insights into the different catalytic activities of CYP3A4 and CYP3A5 toward schisantherin E

“…The residue 481 was replaced by Gly481 in CYP3A4. Paolo et al studied the selectivity of CYP3A4 by changing active site crowding and found that the G481L mutant could alter the selectivity of CYP3A4 (Schiavini, Cheong, Moitessier, & Auclair, 2017).…”