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 Kathleen Boal, Joseph M. Bollinger, Jr.

Research output: Contribution to journalArticle

Abstract

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 O2 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 [O2] slows C21•-O2 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.

Original languageEnglish (US)
Pages (from-to)9964-9979
Number of pages16
JournalJournal of the American Chemical Society
Volume141
Issue number25
DOIs
StatePublished - Jun 26 2019

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Tyrosine
Hydrogen
Atoms
Carbon
Iron
Glutarates
Oxygenases
Hydroxylation
Reducing Agents
Substrates
Phenylalanine
Catalyst supports
Enzymes
Ligands
verruculogen
Oxygen

All Science Journal Classification (ASJC) codes

  • Catalysis
  • Chemistry(all)
  • Biochemistry
  • Colloid and Surface Chemistry

Cite this

@article{cb819f52f9dc4539a90137a8c8f7e7ab,
title = "Hydrogen Donation but not Abstraction by a Tyrosine (Y68) during Endoperoxide Installation by Verruculogen Synthase (FtmOx1)",
abstract = "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 O2 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{\^a}€¢) 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 [O2] slows C21{\^a}€¢-O2 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.",
author = "Dunham, {Noah P.} and {Del R{\'i}o Pantoja}, {Jos{\'e} M.} and Bo Zhang and Rajakovich, {Lauren J.} and Allen, {Benjamin D.} and Carsten Krebs and Boal, {Amie Kathleen} and {Bollinger, Jr.}, {Joseph M.}",
year = "2019",
month = "6",
day = "26",
doi = "10.1021/jacs.9b03567",
language = "English (US)",
volume = "141",
pages = "9964--9979",
journal = "Journal of the American Chemical Society",
issn = "0002-7863",
publisher = "American Chemical Society",
number = "25",

}

Hydrogen Donation but not Abstraction by a Tyrosine (Y68) during Endoperoxide Installation by Verruculogen Synthase (FtmOx1). / Dunham, Noah P.; Del Río Pantoja, José M.; Zhang, Bo; Rajakovich, Lauren J.; Allen, Benjamin D.; Krebs, Carsten; Boal, Amie Kathleen; Bollinger, Jr., Joseph M.

In: Journal of the American Chemical Society, Vol. 141, No. 25, 26.06.2019, p. 9964-9979.

Research output: Contribution to journalArticle

TY - JOUR

T1 - Hydrogen Donation but not Abstraction by a Tyrosine (Y68) during Endoperoxide Installation by Verruculogen Synthase (FtmOx1)

AU - Dunham, Noah P.

AU - Del Río Pantoja, José M.

AU - Zhang, Bo

AU - Rajakovich, Lauren J.

AU - Allen, Benjamin D.

AU - Krebs, Carsten

AU - Boal, Amie Kathleen

AU - Bollinger, Jr., Joseph M.

PY - 2019/6/26

Y1 - 2019/6/26

N2 - 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 O2 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 [O2] slows C21•-O2 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.

AB - 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 O2 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 [O2] slows C21•-O2 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.

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