6OXH

X-ray crystal structure of His-tagged Y140F FtmOx1 bound to Fe(II) and 2-oxoglutarate

Summary for 6OXH

• Entry DOI: 10.2210/pdb6oxh/pdb
• Descriptor: Verruculogen synthase, FE (II) ION, 2-OXOGLUTARIC ACID, ... (4 entities in total)
• Functional Keywords: endoperoxidation, deprenylation, oxygenase, metalloenzyme, fumitremorgin, verruculogen, oxidoreductase
• Biological source: Neosartorya fumigata (strain ATCC MYA-4609 / Af293 / CBS 101355 / FGSC A1100) (Aspergillus fumigatus Af293)
• Total number of polymer chains: 2
• Total formula weight: 70610.23

Authors

Dunham, N.P.,Boal, A.K. (deposition date: 2019-05-13, release date: 2019-07-03, Last modification date: 2023-10-11)

Primary citation

Dunham, N.P.,Del Rio Pantoja, J.M.,Zhang, B.,Rajakovich, L.J.,Allen, B.D.,Krebs, C.,Boal, A.K.,Bollinger Jr., J.M.
Hydrogen Donation but not Abstraction by a Tyrosine (Y68) during Endoperoxide Installation by Verruculogen Synthase (FtmOx1).
J.Am.Chem.Soc., 141:9964-9979, 2019

PubMed 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 O 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] slows C21•-O 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.

PubMed: 31117657
DOI: 10.1021/jacs.9b03567

Experimental method

X-RAY DIFFRACTION (1.92 Å)