Abstract
The acid/base-catalysed Kemp elimination of 5-nitro-benzisoxazole forming 2-cyano-4-nitrophenol has long served as a design platform of enzymes with non-natural reactions, providing new mechanistic insights in protein science. Here we describe an alternative concept based on redox catalysis by P450-BM3, leading to the same Kemp product via a fundamentally different mechanism. QM/MM computations show that it involves coordination of the substrate’s N-atom to haem-Fe(II) with electron transfer and concomitant N–O heterolysis liberating an intermediate having a nitrogen radical moiety Fe(III)–N· and a phenoxyl anion. Product formation occurs by bond rotation and H-transfer. Two rationally chosen point mutations cause a notable increase in activity. The results shed light on the prevailing mechanistic uncertainties in human P450-catalysed metabolism of the immunomodulatory drug leflunomide, which likewise undergoes redox-mediated Kemp elimination by P450-BM3. Other isoxazole-based pharmaceuticals are probably also metabolized by a redox mechanism. Our work provides a basis for designing future artificial enzymes.
Highlights
The acid/base-catalysed Kemp elimination of 5-nitro-benzisoxazole forming 2-cyano-4nitrophenol has long served as a design platform of enzymes with non-natural reactions, providing new mechanistic insights in protein science
To exclude the possibility that an unknown protein in the cell free extract (CFE) may catalyse this Kemp elimination, the enzyme was purified and tested, and the product identity was confirmed by gas chromatography–mass spectrometry (GC–MS) analysis (Supplementary Figs 1 and 2)
Various protein scaffolds have been reported as Kemp eliminases, all operating by the traditional acid/base mechanism[2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17]
Summary
The acid/base-catalysed Kemp elimination of 5-nitro-benzisoxazole forming 2-cyano-4nitrophenol has long served as a design platform of enzymes with non-natural reactions, providing new mechanistic insights in protein science. Combining computational design methodology with 17 rounds of state-of-the-art directed evolution Hilvert, Mayo and coworkers reported the artificial enzyme HG3.17, which employs a Brønsted acid/base mechanism to catalyse Kemp elimination with an unprecedented catalytic efficiency that approaches the activities of natural enzyme (kcat 1⁄4 700 s À 1 and kcat/Km 1⁄4 230,000 M À 1 s À 1)[16]. While these numbers are truly impressive, analysis of the possible efficiency limits for base-catalysed Kemp elimination shows that additional improvement is still possible in principle[17]. From a fundamental point, finding a protein scaffold that catalyses the Kemp elimination by a fundamentally different mechanism would further our understanding of the function, genesis and evolution of enzymes, and provide new opportunities for creating catalysts for novel chemical transformations
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
