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Selective oxidation of carbolide C-H bonds by an engineered macrolide P450 mono-oxygenase.

Li S, Chaulagain MR, Knauff AR, Podust LM, Montgomery J, Sherman DH
Proc Natl Acad Sci U S A 106 18463-8 (2009)
Cited: 57 times
EuropePMC logo PMID: 19833867

Abstract

Regio- and stereoselective oxidation of an unactivated C-H bond remains a central challenge in organic chemistry. Considerable effort has been devoted to identifying transition metal complexes, biological catalysts, or simplified mimics, but limited success has been achieved. Cytochrome P450 mono-oxygenases are involved in diverse types of regio- and stereoselective oxidations, and represent a promising biocatalyst to address this challenge. The application of this class of enzymes is particularly significant if their substrate spectra can be broadened, selectivity controlled, and reactions catalyzed in the absence of expensive heterologous redox partners. In this study, we engineered a macrolide biosynthetic P450 mono-oxygenase PikC (PikC(D50N)-RhFRED) with remarkable substrate flexibility, significantly increased activity compared to wild-type enzyme, and self-sufficiency. By harnessing its unique desosamine-anchoring functionality via a heretofore under-explored "substrate engineering" strategy, we demonstrated the ability of PikC to hydroxylate a series of carbocyclic rings linked to the desosamine glycoside via an acetal linkage (referred to as "carbolides") in a regioselective manner. Complementary analysis of a number of high-resolution enzyme-substrate cocrystal structures provided significant insights into the function of the aminosugar-derived anchoring group for control of reaction site selectivity. Moreover, unexpected biological activity of a select number of these carbolide systems revealed their potential as a previously unrecorded class of antibiotics.

Reviews - 2wi9 mentioned but not cited (2)

  1. Cytochromes P450 for natural product biosynthesis in Streptomyces: sequence, structure, and function. Rudolf JD, Chang CY, Ma M, Shen B. Nat Prod Rep 34 1141-1172 (2017)
  2. Conformational plasticity and structure/function relationships in cytochromes P450. Pochapsky TC, Kazanis S, Dang M. Antioxid Redox Signal 13 1273-1296 (2010)

Articles - 2wi9 mentioned but not cited (2)

  1. Selective oxidation of carbolide C-H bonds by an engineered macrolide P450 mono-oxygenase. Li S, Chaulagain MR, Knauff AR, Podust LM, Montgomery J, Sherman DH. Proc Natl Acad Sci U S A 106 18463-18468 (2009)
  2. Structure-Function Analysis of the Biotechnologically Important Cytochrome P450 107 (CYP107) Enzyme Family. Padayachee T, Lamb DC, Nelson DR, Syed K. Biomolecules 13 1733 (2023)


Reviews citing this publication (14)

  1. Mechanical properties of cellularly responsive hydrogels and their experimental determination. Kloxin AM, Kloxin CJ, Bowman CN, Anseth KS. Adv Mater 22 3484-3494 (2010)
  2. Cytochrome P450: taming a wild type enzyme. Jung ST, Lauchli R, Arnold FH. Curr Opin Biotechnol 22 809-817 (2011)
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  6. Selective CH bond functionalization with engineered heme proteins: new tools to generate complexity. Zhang RK, Huang X, Arnold FH. Curr Opin Chem Biol 49 67-75 (2019)
  7. P450 Monooxygenases Enable Rapid Late-Stage Diversification of Natural Products via C-H Bond Activation. Fessner ND. ChemCatChem 11 2226-2242 (2019)
  8. Engineering and application of P450 monooxygenases in pharmaceutical and metabolite synthesis. Caswell JM, O'Neill M, Taylor SJ, Moody TS. Curr Opin Chem Biol 17 271-275 (2013)
  9. Controlling substrate specificity and product regio- and stereo-selectivities of P450 enzymes without mutagenesis. Polic V, Auclair K. Bioorg Med Chem 22 5547-5554 (2014)
  10. Frontiers and opportunities in chemoenzymatic synthesis. Mortison JD, Sherman DH. J Org Chem 75 7041-7051 (2010)
  11. Streptomyces spp. in the biocatalysis toolbox. Spasic J, Mandic M, Djokic L, Nikodinovic-Runic J. Appl Microbiol Biotechnol 102 3513-3536 (2018)
  12. A Unique P450 Peroxygenase System Facilitated by a Dual-Functional Small Molecule: Concept, Application, and Perspective. Di S, Fan S, Jiang F, Cong Z. Antioxidants (Basel) 11 529 (2022)
  13. Use of chemical auxiliaries to control p450 enzymes for predictable oxidations at unactivated C-h bonds of substrates. Auclair K, Polic V. Adv Exp Med Biol 851 209-228 (2015)
  14. Ru(II)-diimine complexes and cytochrome P450 working hand-in-hand. Eidenschenk C, Cheruzel L. J Inorg Biochem 213 111254 (2020)

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  1. Controlled oxidation of remote sp3 C-H bonds in artemisinin via P450 catalysts with fine-tuned regio- and stereoselectivity. Zhang K, Shafer BM, Demars MD, Stern HA, Fasan R. J Am Chem Soc 134 18695-18704 (2012)
  2. P450-catalyzed intramolecular sp3 C-H amination with arylsulfonyl azide substrates. Singh R, Bordeaux M, Fasan R. ACS Catal 4 546-552 (2014)
  3. Hydrogen peroxide-independent production of α-alkenes by OleTJE P450 fatty acid decarboxylase. Liu Y, Wang C, Yan J, Zhang W, Guan W, Lu X, Li S. Biotechnol Biofuels 7 28 (2014)
  4. Enzymatic hydroxylation of an unactivated methylene C-H bond guided by molecular dynamics simulations. Narayan AR, Jiménez-Osés G, Liu P, Negretti S, Zhao W, Gilbert MM, Ramabhadran RO, Yang YF, Furan LR, Li Z, Podust LM, Montgomery J, Houk KN, Sherman DH. Nat Chem 7 653-660 (2015)
  5. Expanding the toolbox of organic chemists: directed evolution of P450 monooxygenases as catalysts in regio- and stereoselective oxidative hydroxylation. Roiban GD, Reetz MT. Chem Commun (Camb) 51 2208-2224 (2015)
  6. Discovery of potent parthenolide-based antileukemic agents enabled by late-stage P450-mediated C-H functionalization. Kolev JN, O'Dwyer KM, Jordan CT, Fasan R. ACS Chem Biol 9 164-173 (2014)
  7. Anti-Markovnikov alkene oxidation by metal-oxo-mediated enzyme catalysis. Hammer SC, Kubik G, Watkins E, Huang S, Minges H, Arnold FH. Science 358 215-218 (2017)
  8. New reactions and products resulting from alternative interactions between the P450 enzyme and redox partners. Zhang W, Liu Y, Yan J, Cao S, Bai F, Yang Y, Huang S, Yao L, Anzai Y, Kato F, Podust LM, Sherman DH, Li S. J Am Chem Soc 136 3640-3646 (2014)
  9. Substrate recognition by the multifunctional cytochrome P450 MycG in mycinamicin hydroxylation and epoxidation reactions. Li S, Tietz DR, Rutaganira FU, Kells PM, Anzai Y, Kato F, Pochapsky TC, Sherman DH, Podust LM. J Biol Chem 287 37880-37890 (2012)
  10. Directing group-controlled regioselectivity in an enzymatic C-H bond oxygenation. Negretti S, Narayan AR, Chiou KC, Kells PM, Stachowski JL, Hansen DA, Podust LM, Montgomery J, Sherman DH. J Am Chem Soc 136 4901-4904 (2014)
  11. Enhancing the efficiency and regioselectivity of P450 oxidation catalysts by unnatural amino acid mutagenesis. Kolev JN, Zaengle JM, Ravikumar R, Fasan R. Chembiochem 15 1001-1010 (2014)
  12. Chemoenzymatic Total Synthesis and Structural Diversification of Tylactone-Based Macrolide Antibiotics through Late-Stage Polyketide Assembly, Tailoring, and C-H Functionalization. Lowell AN, DeMars MD, Slocum ST, Yu F, Anand K, Chemler JA, Korakavi N, Priessnitz JK, Park SR, Koch AA, Schultz PJ, Sherman DH. J Am Chem Soc 139 7913-7920 (2017)
  13. LICRED: a versatile drop-in vector for rapid generation of redox-self-sufficient cytochrome P450s. Sabbadin F, Hyde R, Robin A, Hilgarth EM, Delenne M, Flitsch S, Turner N, Grogan G, Bruce NC. Chembiochem 11 987-994 (2010)
  14. Biocatalytic synthesis of pikromycin, methymycin, neomethymycin, novamethymycin, and ketomethymycin. Hansen DA, Rath CM, Eisman EB, Narayan AR, Kittendorf JD, Mortison JD, Yoon YJ, Sherman DH. J Am Chem Soc 135 11232-11238 (2013)
  15. Structural evidence for enhancement of sequential vitamin D3 hydroxylation activities by directed evolution of cytochrome P450 vitamin D3 hydroxylase. Yasutake Y, Fujii Y, Nishioka T, Cheon WK, Arisawa A, Tamura T. J Biol Chem 285 31193-31201 (2010)
  16. Characterization of a Unique Pathway for 4-Cresol Catabolism Initiated by Phosphorylation in Corynebacterium glutamicum. Du L, Ma L, Qi F, Zheng X, Jiang C, Li A, Wan X, Liu SJ, Li S. J Biol Chem 291 6583-6594 (2016)
  17. An efficient route to selective bio-oxidation catalysts: an iterative approach comprising modeling, diversification, and screening, based on CYP102A1. Seifert A, Antonovici M, Hauer B, Pleiss J. Chembiochem 12 1346-1351 (2011)
  18. Characterization of Heronamide Biosynthesis Reveals a Tailoring Hydroxylase and Indicates Migrated Double Bonds. Zhu Y, Zhang W, Chen Y, Yuan C, Zhang H, Zhang G, Ma L, Zhang Q, Tian X, Zhang S, Zhang C. Chembiochem 16 2086-2093 (2015)
  19. Acyl-CoA subunit selectivity in the pikromycin polyketide synthase PikAIV: steady-state kinetics and active-site occupancy analysis by FTICR-MS. Bonnett SA, Rath CM, Shareef AR, Joels JR, Chemler JA, Håkansson K, Reynolds K, Sherman DH. Chem Biol 18 1075-1081 (2011)
  20. Engineering of versatile redox partner fusions that support monooxygenase activity of functionally diverse cytochrome P450s. Bakkes PJ, Riehm JL, Sagadin T, Rühlmann A, Schubert P, Biemann S, Girhard M, Hutter MC, Bernhardt R, Urlacher VB. Sci Rep 7 9570 (2017)
  21. Nickel-Catalyzed Regiodivergent Approach to Macrolide Motifs. Shareef AR, Sherman DH, Montgomery J. Chem Sci 3 892-895 (2012)
  22. Cosubstrate tolerance of the aminoglycoside resistance enzyme Eis from Mycobacterium tuberculosis. Chen W, Green KD, Garneau-Tsodikova S. Antimicrob Agents Chemother 56 5831-5838 (2012)
  23. Strategies for Substrate-Regulated P450 Catalysis: From Substrate Engineering to Co-catalysis. Xu J, Wang C, Cong Z. Chemistry 25 6853-6863 (2019)
  24. A new structural form in the SAM/metal-dependent o‑methyltransferase family: MycE from the mycinamicin biosynthetic pathway. Akey DL, Li S, Konwerski JR, Confer LA, Bernard SM, Anzai Y, Kato F, Sherman DH, Smith JL. J Mol Biol 413 438-450 (2011)
  25. Production of pikromycin using branched chain amino acid catabolism in Streptomyces venezuelae ATCC 15439. Yi JS, Kim M, Kim EJ, Kim BG. J Ind Microbiol Biotechnol 45 293-303 (2018)
  26. The crystal structure of the versatile cytochrome P450 enzyme CYP109B1 from Bacillus subtilis. Zhang A, Zhang T, Hall EA, Hutchinson S, Cryle MJ, Wong LL, Zhou W, Bell SG. Mol Biosyst 11 869-881 (2015)
  27. Synthesis of Diverse 11- and 12-Membered Macrolactones from a Common Linear Substrate Using a Single Biocatalyst. Gilbert MM, DeMars MD, Yang S, Grandner JM, Wang S, Wang H, Narayan ARH, Sherman DH, Houk KN, Montgomery J. ACS Cent Sci 3 1304-1310 (2017)
  28. Biochemical and Structural Characterization of MycCI, a Versatile P450 Biocatalyst from the Mycinamicin Biosynthetic Pathway. DeMars MD, Sheng F, Park SR, Lowell AN, Podust LM, Sherman DH. ACS Chem Biol 11 2642-2654 (2016)
  29. An artificial self-sufficient cytochrome P450 directly nitrates fluorinated tryptophan analogs with a different regio-selectivity. Zuo R, Zhang Y, Huguet-Tapia JC, Mehta M, Dedic E, Bruner SD, Loria R, Ding Y. Biotechnol J 11 624-632 (2016)
  30. Chemoenzymatic Route to Oxyfunctionalized Cembranoids Facilitated by Substrate and Protein Engineering. Le-Huu P, Rekow D, Krüger C, Bokel A, Heidt T, Schaubach S, Claasen B, Hölzel S, Frey W, Laschat S, Urlacher VB. Chemistry 24 12010-12021 (2018)
  31. Selective oxidation of aliphatic C-H bonds in alkylphenols by a chemomimetic biocatalytic system. Du L, Dong S, Zhang X, Jiang C, Chen J, Yao L, Wang X, Wan X, Liu X, Wang X, Huang S, Cui Q, Feng Y, Liu SJ, Li S. Proc Natl Acad Sci U S A 114 E5129-E5137 (2017)
  32. A high-throughput screen for the engineered production of β-lactam antibiotics. Phelan RM, DiPardo BJ, Townsend CA. ACS Chem Biol 7 835-840 (2012)
  33. Cytochrome P450Blt Enables Versatile Peptide Cyclisation to Generate Histidine- and Tyrosine-Containing Crosslinked Tripeptide Building Blocks. Zhao Y, Marschall E, Treisman M, McKay A, Padva L, Crüsemann M, Nelson DR, Steer DL, Schittenhelm RB, Tailhades J, Cryle MJ. Angew Chem Int Ed Engl 61 e202204957 (2022)
  34. Enzymatic Late-Stage Oxidation of Lead Compounds with Solubilizing Biomimetic Docking/Protecting groups. Vickers C, Backfisch G, Oellien F, Piel I, Lange UEW. Chemistry 24 17936-17947 (2018)
  35. Molecular Basis of Iterative C─H Oxidation by TamI, a Multifunctional P450 monooxygenase from the Tirandamycin Biosynthetic Pathway. Newmister SA, Srivastava KR, Espinoza RV, Haatveit KC, Khatri Y, Martini RM, Garcia-Borràs M, Podust LM, Houk KN, Sherman DH. ACS Catal 10 13445-13454 (2020)
  36. Positioning-Group-Enabled Biocatalytic Oxidative Dearomatization. Dockrey SAB, Suh CE, Benítez AR, Wymore T, Brooks CL, Narayan ARH. ACS Cent Sci 5 1010-1016 (2019)
  37. Selective biocatalytic hydroxylation of unactivated methylene C-H bonds in cyclic alkyl substrates. Sarkar MR, Dasgupta S, Pyke SM, Bell SG. Chem Commun (Camb) 55 5029-5032 (2019)
  38. The complete genome sequence of the nitrile biocatalyst Rhodocccus rhodochrous ATCC BAA-870. Frederick J, Hennessy F, Horn U, de la Torre Cortés P, van den Broek M, Strych U, Willson R, Hefer CA, Daran JG, Sewell T, Otten LG, Brady D. BMC Genomics 21 3 (2020)
  39. Unnatural activities and mechanistic insights of cytochrome P450 PikC gained from site-specific mutagenesis by non-canonical amino acids. Pan Y, Li G, Liu R, Guo J, Liu Y, Liu M, Zhang X, Chi L, Xu K, Wu R, Zhang Y, Li Y, Gao X, Li S. Nat Commun 14 1669 (2023)


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