Structural insights into the functional convergence of two divergent cytochrome P450 enzymes
-
GRAPHICAL ABSTRACT
DownLoad:
Full size image
-
PUBLIC SUMMARY
-
Cytochrome P450 enzymes are powerful biocatalysts for selective C–H bond oxidation.
Functional convergence of divergent P450s enable regio- and stereoselective oxidation of the same substrate.
Catalytic pocket geometry and polar residues govern P450 substrate orientation and catalytic selectivity.
-
-
ABSTRACT
Cytochrome P450 enzymes are versatile biocatalysts for a broad spectrum of oxidative reactions and a hotspot for protein engineering. Using the “multi-enzymes-for-multi-substrates” directed evolution strategy, we have found a number of mutants of the two archetypal P450 enzymes (i.e., P450cam and P450BM3) to catalyze the regio- and stereoselective oxidation of the perfume compound (–)-ambroxide. Despite their low sequence similarity, these mutant enzymes exhibit a convergent activity of 3β-hydroxylation as a result of laboratory-directed evolution. To address the longstanding interest in elucidating the principle underlying substrate recognition and catalytic specificity of P450 enzymes, herein, we resolve the crystal structures of P450cam-F87R and P450BM3-F87A/L75N/V78S (heme domain) in complex with (–)-ambroxide. Comparative analysis reveals how specific amino acid mutations reshape the substrate-binding pocket, thereby enhancing substrate accommodation and catalytic precision. The introduction of polar residues in the catalytic pocket is essential for controlling the optimal orientation of the substrate with a heterocycle. These findings underscore the importance of the pocket shape and polarity in enzyme redesign and provide valuable insights for the (semi-)rational engineering of P450 enzymes. -
-
REFERENCES
[1] Guillemard L., Kaplaneris N., Ackermann L., et al. (2021). Late-stage C−H functionalization offers new opportunities in drug discovery. Nat. Rev. Chem. 5:522−545. DOI:10.1038/s41570-021-00300-6 [2] McIntosh J. A., Farwell C. C. and Arnold F. H. (2014). Expanding P450 catalytic reaction space through evolution and engineering. Curr. Opin. Chem. Biol. 19:126−134. DOI:10.1016/j.cbpa.2014.02.001 [3] Xu H.-J., Fan Z., Nian B.-B., et al. (2025). Achieving mono-selective palladium(II)-catalysed C–H activation of arenes with protein ligands. Nat. Catal. 8:948−956. DOI:10.1038/s41929-025-01407-5 [4] Sener C., Timokhin V. I., Hellinger J., et al. (2025). Pd/C promotes C−H bond activation and oxidation of p-hydroxybenzoate during hydrogenolysis of poplar. Nat. Commun. 16:5259. DOI:10.1038/s41467-025-60270-x [5] Song F., Zheng M., Wang J., et al. (2023). Chemoenzymatic synthesis of C14-functionalized steroids. Nat. Synth. 2:729−739. DOI:10.1038/s44160-023-00280-z [6] Craven E. J., Latham J., Shepherd S. A., et al. (2021). Programmable late-stage C−H bond functionalization enabled by integration of enzymes with chemocatalysis. Nat. Catal. 4:385−394. DOI:10.1038/s41929-021-00603-3 [7] Zhang X. and Li S. (2017). Expansion of chemical space for natural products by uncommon P450 reactions. Nat. Prod. Rep. 34:1061−1089. DOI:10.1039/c7np00028f [8] Zhang X., Guo J., Cheng F., et al. (2021). Cytochrome P450 enzymes in fungal natural product biosynthesis. Nat. Prod. Rep. 38:1072−1099. DOI:10.1039/d1np00004g [9] Bernhardt R. (2006). Cytochromes P450 as versatile biocatalysts. J. Biotechnol. 124:128−145. DOI:10.1016/j.jbiotec.2006.01.026 [10] Bernhardt R. and Urlacher V. B. (2014). Cytochromes P450 as promising catalysts for biotechnological application: Chances and limitations. Appl. Microbiol. Biotechnol. 98:6185−6203. DOI:10.1007/s00253-014-5767-7 [11] Ma L., Sun T., Liu Y., et al. (2023). Enzymatic synthesis of indigo derivatives by tuning P450 BM3 peroxygenases. Synth. Syst. Biotechnol. 8:452−461. DOI:10.1016/j.synbio.2023.06.006 [12] Sirim D., Widmann M., Wagner F., et al. (2010). Prediction and analysis of the modular structure of cytochrome P450 monooxygenases. BMC Struct. Biol. 10:34. DOI:10.1186/1472-6807-10-34 [13] Hamdane D., Zhang H. and Hollenberg P. (2008). Oxygen activation by cytochrome P450 monooxygenase. Photosynth. Res. 98:657−666. DOI:10.1007/s11120-008-9322-1 [14] Whitehouse C. J., Bell S. G. and Wong L. L. (2012). P450(BM3) (CYP102A1): Connecting the dots. Chem. Soc. Rev. 41:1218−1260. DOI:10.1039/c1cs15192d [15] Schlichting I., Berendzen J., Chu K., et al. (2000). The catalytic pathway of cytochrome P450cam at atomic resolution. Science 287:1615−1622. DOI:10.1126/science.287.5458.1615 [16] Gunsalus I. C. and Wagner G. C. (1978). Bacterial P-450cam methylene monooxygenase components: Cytochrome m, putidaredoxin, and putidaredoxin reductase. Methods Enzymol. 52:166−188. DOI:10.1016/s0076-6879(78)52019-3 [17] Ahalawat N. and Mondal J. (2018). Mapping the substrate recognition pathway in cytochrome P450. J. Am. Chem. Soc. 140:17743−17752. DOI:10.1021/jacs.8b10840 [18] Follmer A. H., Mahomed M., Goodin D. B., et al. (2018). Substrate-dependent allosteric regulation in cytochrome P450cam (CYP101A1). J. Am. Chem. Soc. 140:16222−16228. DOI:10.1021/jacs.8b09441 [19] Sevrioukova I. F., Poulos T. L. and Churbanova I. Y. (2010). Crystal structure of the putidaredoxin reductase x putidaredoxin electron transfer complex. J. Biol. Chem. 285:13616−13620. DOI:10.1074/jbc.M110.104968 [20] Hollingsworth S. A., Batabyal D., Nguyen B. D., et al. (2016). Conformational selectivity in cytochrome P450 redox partner interactions. Proc. Natl. Acad. Sci. USA 113:8723−8728. DOI:10.1073/pnas.1606474113 [21] Hudeeek J., Baumruk V., Anzenbacher P., et al. (1998). Catalytically self-sufficient P450 CYP102 (cytochrome P450 BM-3): Resonance Raman spectral characterization of the heme domain and of the holoenzyme. Biochem. Biophys. Res. Commun. 243:811−815. DOI:10.1006/bbrc.1997.8057 [22] Ma L., Li F. W., Zhang X. W., et al. (2022). Development of MEMS directed evolution strategy for multiplied throughput and convergent evolution of cytochrome P450 enzymes. Sci. China Life Sci. 65:550−560. DOI:10.1007/s11427-021-1994-1 [23] Xue Y. Q., Wilson D., Zhao L. S., et al. (1998). Hydroxylation of macrolactones YC-17 and narbomycin is mediated by the pikC-encoded cytochrome P450 in Streptomyces venezuelae. Chem. Biol. 5:661−667. DOI:10.1016/S1074-5521(98)90293-9 [24] Du L., Dong S., Zhang X. W., et al. (2017). Selective oxidation of aliphatic C−H bonds in alkylphenols by a chemomimetic biocatalytic system. Proc. Natl. Acad. Sci. USA 114:E5129−E5137. DOI:10.1073/pnas.1702317114 [25] Boddupalli S. S., Estabrook R. W. and Peterson J. A. (1990). Fatty acid monooxygenation by cytochrome P-450BM-3. J. Biol. Chem. 265: 4233-4239. https://www.jbc.org/article/S0021-9258(19)39552-3/fulltext [26] Poulos T. L., Finzel B. C. and Howard A. J. (1987). High-resolution crystal structure of cytochrome P450cam. J. Mol. Biol. 195:687−700. DOI:10.1016/0022-2836(87)90190-2 [27] Guengerich F. P., Martin M. V., Sohl C. D., et al. (2009). Measurement of cytochrome P450 and NADPH-cytochrome P450 reductase. Nat. Protoc. 4:1245−1251. DOI:10.1038/nprot.2009.121 [28] Panicco P., Astuti Y., Fantuzzi A., et al. (2008). P450 versus P420: Correlation between cyclic voltammetry and visible absorption spectroscopy of the immobilized heme domain of cytochrome P450 BM3. J. Phys. Chem. B 112:14063−14068. DOI:10.1021/jp8050033 [29] Sevrioukova I. F. and Poulos T. L. (2002). Putidaredoxin reductase, a new function for an old protein. J. Biol. Chem. 277:25831−25839. DOI:10.1074/jbc.M201110200 [30] Li S., Podust L. M. and Sherman D. H. (2007). Engineering and analysis of a self-sufficient biosynthetic cytochrome P450 PikC fused to the RhFRED reductase domain. J. Am. Chem. Soc. 129:12940−12941. DOI:10.1021/ja075842d [31] Xu Q., Kong H. T., Liu K., et al. (2023). The biosafety level-2 macromolecular crystallography beamline (BL10U2) at the Shanghai Synchrotron Radiation Facility. Nucl. Sci. Tech. 34:202. DOI:10.1007/s41365-023-01350-9 [32] Xiao Q., Wu T., Bao K., et al. (2024). Upgrade of crystallography beamline BL19U1 at the Shanghai Synchrotron Radiation Facility. J. Appl. Crystallogr. 57:630−637. DOI:10.1107/S1600576724002188 [33] Kabsch W. (2010). Xds. Acta. Crystallogr. D Biol. Crystallogr. 66:125−132. DOI:10.1107/S0907444909047337 [34] Nagano S. and Poulos T. L. (2005). Crystallographic study on the dioxygen complex of wild-type and mutant cytochrome P450cam. Implications for the dioxygen activation mechanism. J. Biol. Chem. 280:31659−31663. DOI:10.1074/jbc.M505261200 [35] Hegde A., Haines D. C., Bondlela M., et al. (2007). Interactions of substrates at the surface of P450s can greatly enhance substrate potency. Biochemistry 46:14010−14017. DOI:10.1021/bi701667m [36] Adams P. D., Afonine P. V., Bunkoczi G., et al. (2010). PHENIX: A comprehensive Python-based system for macromolecular structure solution. Acta. Crystallogr. D Biol. Crystallogr. 66:213−221. DOI:10.1107/S0907444909052925 [37] Emsley P., Lohkamp B., Scott W. G., et al. (2010). Features and development of Coot. Acta. Crystallogr. D Biol. Crystallogr. 66:486−501. DOI:10.1107/S0907444910007493 [38] Li S., Ouellet H., Sherman D. H., et al. (2009). Analysis of transient and catalytic desosamine-binding pockets in cytochrome P-450 PikC from Streptomyces venezuelae. J. Biol. Chem. 284:5723−5730. DOI:10.1074/jbc.M807592200 [39] Zhang X., Jiang Y., Chen Q., et al. (2021). H-bonding networks dictate the molecular mechanism of H2O2 activation by P450. ACS Catal. 11:8774−8785. DOI:10.1021/acscatal.1c02068 [40] Chen J., Dong S., Fang W., et al. (2023). Regiodivergent and enantioselective hydroxylation of C−H bonds by synergistic use of protein engineering and exogenous dual-functional small molecules. Angew. Chem. Int. Ed. Engl. 62:e202215088. DOI:10.1002/anie.202215088 [41] Ma N. N., Chen Z. F., Chen J., et al. (2018). Dual-functional small molecules for generating an efficient cytochrome P450BM3 peroxygenase. Angew. Chem. Int. Edit. 57:7628−7633. DOI:10.1002/anie.201801592 [42] Kong F. H., Chen J., Qin X. Q., et al. (2022). Evolving a P450BM3 peroxygenase for the production of indigoid dyes from indoles. Chemcatchem 14:e202201151. DOI:10.1002/cctc.202201151 [43] Fansher D. J., Besna J. N., Fendri A., et al. (2024). Choose your own adventure: A comprehensive database of reactions catalyzed by cytochrome P450 BM3 variants. ACS Catal. 14:5560−5592. DOI:10.1021/acscatal.4c00086 [44] Hoffmann G., Bonsch K., Greiner-Stoffele T., et al. (2011). Changing the substrate specificity of P450cam towards diphenylmethane by semi-rational enzyme engineering. Protein Eng. Des. Sel. 24:439−446. DOI:10.1093/protein/gzq119 [45] Ekici O. D., Paetzel M. and Dalbey R. E. (2008). Unconventional serine proteases: Variations on the catalytic Ser/His/Asp triad configuration. Protein Sci. 17:2023−2037. DOI:10.1110/ps.035436.108 [46] Galperin M. Y. and Koonin E. V. (2012). Divergence and convergence in enzyme evolution. J. Biol. Chem. 287:21−28. DOI:10.1074/jbc.R111.241976 [47] Cupp-Vickery J. R., Han O., Hutchinson C. R., et al. (1996). Substrate-assisted catalysis in cytochrome P450eryF. Nat. Struct. Biol. 3:632−637. DOI:10.1038/nsb0796-632 -
ABOUT THIS ARTICLE
Cite this article:
Ma L., Dong S., Zhang J., et al. (2026). Structural insights into the functional convergence of two divergent cytochrome P450 enzymes. The Innovation Life 4:100221. https://doi.org/10.59717/j.xinn-life.2026.100221
Welcome!
To request copyright permission to republish or share portions of our works, please visit Copyright Clearance Center's (CCC) Marketplace website at marketplace.copyright.com.
Export File
Citation
Ma L., Dong S., Zhang J., et al. (2026). Structural insights into the functional convergence of two divergent cytochrome P450 enzymes. The Innovation Life 4:100221. https://doi.org/10.59717/j.xinn-life.2026.100221
Format
Content
-
Figure 1.
Schematic oxidative reactions of (–)-ambroxide
-
Figure 2.
Product distribution of wild-type and mutant P450 enzymes
-
Figure 3.
Structural analysis of the substrate-binding pockets in P450 variants
-
Figure 4.
Structural basis for the regio- and stereoselectivity of P450 variants toward (–)-ambroxide
-
Figure 5.
Comparative analysis of the substrate binding pockets of P450 variants
-
Figure 6.
QM/MM-derived mechanisms for the regio- and stereoselective hydroxylation of (–)-ambroxide catalyzed by the two P450 mutants
