Engineering C–C Bond Cleavage Activity into a P450 Monooxygenase Enzyme

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dc.contributor.authorMiller, J.C.
dc.contributor.authorLee, J.H.Z.
dc.contributor.authorMclean, M.A.
dc.contributor.authorChao, R.R.
dc.contributor.authorStone, I.S.J.
dc.contributor.authorPukala, T.L.
dc.contributor.authorBruning, J.B.
dc.contributor.authorDe Voss, J.J.
dc.contributor.authorSchuler, M.A.
dc.contributor.authorSligar, S.G.
dc.contributor.authorBell, S.G.
dc.date.issued2023
dc.description.abstractThe cytochrome P450 (CYP) superfamily of heme monooxygenases has demonstrated ability to facilitate hydroxylation, desaturation, sulfoxidation, epoxidation, heteroatom dealkylation, and carbon−carbon bond formation and cleavage (lyase) reactions. Seeking to study the carbon− carbon cleavage reaction of α-hydroxy ketones in mechanistic detail using a microbial P450, we synthesized α-hydroxy ketone probes based on the physiological substrate for a well-characterized benzoic acid metabolizing P450, CYP199A4. After observing low activity with wild-type CYP199A4, subsequent assays with an F182L mutant demonstrated enzyme-dependent C−C bond cleavage toward one of the α-hydroxy ketones. This C−C cleavage reaction was subject to an inverse kinetic solvent isotope effect analogous to that observed in the lyase activity of the human P450 CYP17A1, suggesting the involvement of a species earlier than Compound I in the catalytic cycle. Co-crystallization of F182L-CYP199A4 with this α-hydroxy ketone showed that the substrate bound in the active site with a preference for the (S)-enantiomer in a position which could mimic the topology of the lyase reaction in CYP17A1. Molecular dynamics simulations with an oxy-ferrous model of CYP199A4 revealed a displacement of the substrate to allow for oxygen binding and the formation of the lyase transition state proposed for CYP17A1. This demonstration that a correctly positioned α-hydroxy ketone substrate can realize lyase activity with an unusual inverse solvent isotope effect in an engineered microbial system opens the door for further detailed biophysical and structural characterization of CYP catalytic intermediates.
dc.description.statementofresponsibilityJustin C. Miller, Joel H. Z. Lee, Mark A. Mclean, Rebecca R. Chao, Isobella S. J. Stone, Tara L. Pukala, John B. Bruning, James J. De Voss, Mary A. Schuler, Stephen G. Sligar, and Stephen G. Bell
dc.identifier.citationJournal of the American Chemical Society, 2023; 145(16):9207-9222
dc.identifier.doi10.1021/jacs.3c01456
dc.identifier.issn0002-7863
dc.identifier.issn1520-5126
dc.identifier.orcidLee, J.H.Z. [0000-0001-6288-9034]
dc.identifier.orcidStone, I.S.J. [0000-0002-9474-4961]
dc.identifier.orcidPukala, T.L. [0000-0001-7391-1436]
dc.identifier.orcidBruning, J.B. [0000-0002-6919-1824]
dc.identifier.orcidBell, S.G. [0000-0002-7457-9727]
dc.identifier.urihttps://hdl.handle.net/2440/138175
dc.language.isoen
dc.publisherAmerican Chemical Society (ACS)
dc.relation.granthttp://purl.org/au-research/grants/arc/DP140103229
dc.relation.granthttp://purl.org/au-research/grants/arc/FT140100355
dc.rights© 2023 American Chemical Society
dc.source.urihttps://doi.org/10.1021/jacs.3c01456
dc.subjectHumans
dc.subjectLyases
dc.subjectCatalytic Domain
dc.subjectCatalysis
dc.subjectMolecular Dynamics Simulation
dc.titleEngineering C–C Bond Cleavage Activity into a P450 Monooxygenase Enzyme
dc.typeJournal article
pubs.publication-statusPublished

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