Structural Insight into the Catalytic Mechanism of the Endoperoxide Synthase FtmOx1

Abstract

The 2-oxoglutarate (2OG)-dependent non-heme enzyme FtmOx1 catalyzes the endoperoxide biosynthesis of verruculogen. Although several mechanistic studies have been carried out, the catalytic mechanism of FtmOx1 is not well determined owingtothe lackofareliable complex structure of FtmOx1 with fumitremorgin B. Herein we providet he X-ray crystal structure of the ternary complex FtmOx1•2OG•fumitremorgin Bataresolution of 1.22 .Our structures showthat the binding of fumitremorgin Bi nduces significant compression of the active pocket and that Y68 is in close proximityt o C26 of the substrate.F urther MD simulation and QM/MM calculations support aCarC-like mechanism, in which Y68 acts as the Hatom donor for quenching the C26-centered substrate radical. Our results are consistent with all available experimental data and highlight the importance of accurate complex structures in the mechanistic study of enzymatic catalysis.

Use of Non-canonical Tyrosine Analogs to Probe Control of Radical Intermediates during Endoperoxide Installation by Verruculogen Synthase (FtmOx1)

“…These observations with non-canonical variants thus definitively establish that Tyr68 rather than Tyr224 is the H• donor in the FtmOx1 reaction, echoing the conclusions reached from investigation of the Y68F and Y224F variants 10 and, more recently, from the structure of the FtmOx1•Fe(II)•2OG•1 complex (Figures S10A and S11). 4 The new data are consistent with and rationalize the prior observation that the Y224F variant is fully competent to produce 2 but the Y68F generates an alternative primary product, identified in experiments discussed below.…”

“…The recently published structure of the FtmOx1•Fe(II)•2OG•1 quaternary complex reveals sufficient proximity (4.6 Å) between Fe(II) and C21 to enable direct hydrogen abstraction by the ferryl moiety (Figures S10A). 4 This structure implies that, associated with binding of 1, Tyr224 unexpectedly twists away from Fe(II) (Figure S1).…”

“…1 Each case of enzymatic endoperoxide installation that has been studied to date involves capture of O2 between a carbon radical and nearby olefin of the substrate. [2][3][4] The radical forms by hydrogen atom (H•) transfer (HAT) from the substrate to an oxidized enzyme intermediate generated from or by an iron cofactor. The longest-known and most extensively studied endoperoxide-installing enzymes are the cyclooxygenases (COX), which convert arachidonic acid to PGG2 and PGH2 2 and are targets of nonsteroidal anti-inflammatory drugs.…”

“…5 Two more recently identified endoperoxide synthases belong to the iron(II)-and 2-oxoglutarate-dependent (Fe/2OG) enzyme family. 3,4,6,7 Verruculogen synthase [also known as fumitremorgin B (ftnB) endoperoxidase or FtmOx1; EC 1.14.11.38] from Aspergillus fumigatus installs an endoperoxide between carbons 21 and 27 of ftnB (1 in Figure 1A), producing verruculogen (2 in Figure 1A) on the pathway to the tremor-inducing mycotoxin, fumitremorgin A. 3 NvfI from Aspergillus novofumigatus IBT 16806 converts its substrate asnovolin A to fumigatonoid A by installing both (i) an endoperoxide between methyl (C13) and olefinic (C2') carbons and (ii) a hydroxyl group on C3'reportedly in a single step.…”

“…Very recently, while the study presented here was in preparation, an x-ray crystal structure of the FtmOx1•Fe(II)•2OG•1 complex was finally reported. 4 Although its findings -including (1) proximity of C21 to the cofactor (4.6 Å), (2) proximity of the Tyr68 phenolic oxygen (O 7 ) to C26 (4.0 Å), and (3) greater separation of Tyr224 O 7 from the cofactor (8.5 Å) than in the prior structures of the FtmOx1•Fe(II)•2OG ternary complex (4.2 Å) 3,4,10 are obviously more consistent with the CarC-like mechanism of the second study than with the COX-like mechanism of the first, the structural work provided no direct evidence for the sites of the pair of Tyr radicals observed in the transient-state kinetics experiments. 3 The facial triad, iron(II), and 2OG are shown in orange, and the two contentious Tyr residues that were proposed to be critical for catalysis are highlighted in yellow.…”