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Hydrogen Donation but not Abstraction by a Tyrosine (Y68) During Endoperoxide Installation by Verruculogen Synthase (FtmOx1)

Dunham, Noah P. and Del Río Pantoja, José M. and Zhang, Bo and Rajakovich, Lauren J. and Allen, Benjamin D. and Krebs, Carsten and Boal, Amie K. and Bollinger, J. Martin, Jr. (2019) Hydrogen Donation but not Abstraction by a Tyrosine (Y68) During Endoperoxide Installation by Verruculogen Synthase (FtmOx1). Journal of the American Chemical Society, 141 (25). pp. 9964-9979. ISSN 0002-7863.

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Hydrogen-atom transfer (HAT) from a substrate carbon to an iron(IV)-oxo (ferryl) intermediate initiates a diverse array of enzymatic transformations. For outcomes other than hydroxylation, coupling of the resultant carbon radical and hydroxo ligand (oxygen rebound) must generally be averted. A recent study of FtmOx1, a fungal iron(II)- and 2-(oxo)glutarate-dependent oxygenase that installs the endoperoxide of verruculogen by adding O_2 between carbons 21 and 27 of fumitremorgin B, posited that tyrosine (Tyr or Y) 224 serves as HAT intermediary to separate the C21 radical (C21•) and Fe(III)–OH HAT products and prevent rebound. Our reinvestigation of the FtmOx1 mechanism revealed, instead, direct HAT from C21 to the ferryl complex and surprisingly competitive rebound. The C21-hydroxylated (rebound) product, which undergoes deprenylation, predominates when low [O_2] slows C21•–O_2 coupling in the next step of the endoperoxidation pathway. This pathway culminates with addition of the C21–O–O• peroxyl adduct to olefinic C27 followed by HAT to the C26• from a Tyr. The last step results in sequential accumulation of Tyr radicals, which are suppressed without detriment to turnover by inclusion of the reductant, ascorbate. Replacement of each of four candidates for the proximal C26 H• donor (including Y224) with phenylalanine (F) revealed that only the Y68F variant (i) fails to accumulate the first Tyr• and (ii) makes an altered major product, identifying Y68 as the donor. The implied proximities of C21 to the iron cofactor and C26 to Y68 support a new docking model of the enzyme–substrate complex that is consistent with all available data.

Item Type:Article
Related URLs:
URLURL TypeDescription
Dunham, Noah P.0000-0001-8006-9566
Del Río Pantoja, José M.0000-0002-7989-171X
Zhang, Bo0000-0003-0212-8879
Rajakovich, Lauren J.0000-0001-5412-1981
Allen, Benjamin D.0000-0001-6914-5572
Krebs, Carsten0000-0002-3302-7053
Boal, Amie K.0000-0002-1234-8472
Bollinger, J. Martin, Jr.0000-0003-0751-8585
Additional Information:© 2019 American Chemical Society. Received: April 2, 2019; Published: May 22, 2019. This work was supported by grants from the Searle Scholars Program (to A.K.B.) and the NIH (GM113106 to J.M.B./C.K., GM127079 to C.K., and GM119707 to A.K.B.). Portions of this work were conducted at the Advanced Photon Source (APS), a U.S. Department of Energy (DOE) Office of Science User Facility operated for the DOE Office of Science by Argonne National Laboratory under Contract No. DE-AC02-06CH11357. GM/CA at APS has been funded in whole or in part with Federal funds from the National Cancer Institute (ACB-12002) and the National Institute of General Medical Sciences (AGM-12006). The Eiger 16M detector was funded by the NIH-Office of Research Infrastructure Programs, High-End Instrumentation Grant (1S10OD012289-01A1). Use of LS-CAT Sector 21 was supported by the Michigan Economic Development Corporation and the Michigan Technology Tri-Corridor Grant (085P1000817). We thank Alexey Silakov for advice and technical assistance in the EPR experiments. We also thank the Penn State Proteomics and Mass Spectrometry Facility (University Park, PA). The authors declare no competing financial interest.
Funding AgencyGrant Number
Searle Scholars ProgramUNSPECIFIED
Department of Energy (DOE)DE-AC02-06CH11357
Michigan Economic Development CorporationUNSPECIFIED
Michigan Technology Tri-Corridor Grant085P1000817
National Institute of General Medical SciencesUNSPECIFIED
National Cancer InstituteUNSPECIFIED
Issue or Number:25
Record Number:CaltechAUTHORS:20190523-093737479
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Official Citation:Hydrogen Donation but not Abstraction by a Tyrosine (Y68) during Endoperoxide Installation by Verruculogen Synthase (FtmOx1). Noah P. Dunham, José M. Del Río Pantoja, Bo Zhang, Lauren J. Rajakovich, Benjamin D. Allen, Carsten Krebs, Amie K. Boal, and J. Martin Bollinger, Jr. Journal of the American Chemical Society 2019 141 (25), 9964-9979. DOI: 10.1021/jacs.9b03567
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:95730
Deposited By: Tony Diaz
Deposited On:23 May 2019 17:18
Last Modified:03 Oct 2019 21:16

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