An ENDOR and HYSCORE Investigation of a Reaction Intermediate in IspG (GcpE) Catalysis

Abstract

IspG is a 4Fe-4S protein that carries out an essential reduction step in isoprenoid biosynthesis. Using electron-nuclear double resonance (ENDOR) and hyperfine sublevel correlation (HYSCORE) spectroscopies on labeled samples, we specifically assign the hyperfine interactions in a reaction intermediate. These results help clarify the nature of the reaction intermediate, supporting a direct interaction between the unique 4th Fe in the cluster and the C2 and O3 of the ligand.

Structure, function and inhibition of the two- and three-domain 4Fe-4S IspG proteins

“…We also find that the 1 H ENDOR spectrum of MEcPP bound to AtIspG (Fig. 4A) is essentially identical to that of MEcPP bound to E. coli IspG (2), with an approximately 12 MHz 1 H hyperfine interaction for one of the C2′ methyl protons, proposed elsewhere to be located in a ferraoxetane ring (16). Likewise, the 13 C HYSCORE spectra of [U-13 C]-MEcPP bound to both TtIspG chimeras (Fig.…”

“…Based on the SCORECONS (11) conservation score ranking (Table S1), these residues are ranked as 20, 17, and 10 in terms of their conservation in a series of 310 IspG proteins. We then used Glide followed by molecular mechanics to generate docked poses of the putative ferraoxetane intermediate X (16), as well as of the bound HMBPP product, Fig. S2 B and C. With X, binding was to R55, R101, and N103; with HMBPP, binding was to R55, R101, R128, R232, and K176, residues that are ranked 19, 20, 11, 10, and 17 in terms of their conservation score (Table S1).…”

“…Mechanistically then, it appears that MEcPP binds to the conserved Arg/Lys residues in the A domain and that the carbocation that forms on ring opening is located very close to the 4Fe4S cluster domain, which then leads to formation of the reaction intermediate, X (2,16 (18), and an approximately 17 MHz hyperfine coupling for the C2 carbon (16). To see to what extent it might be possible to predict these spectroscopic observables, we used density functional theory (DFT) (19,20).…”

Isoprenoid biosynthesis in bacterial pathogens

“…The crystal structure for IspH is known for both E. coli (Gräwert et al, 2010) and A. aeolicus (Rekittke et al, 2008). Pyridine-based inhibitors of IspH have undergone spectroscopic and chemical investigation to elucidate their inhibitor functions (Wang et al, 2011b). Indeed, Wang et al (2010) screened a series of small molecules using EPR spectroscopy and put forward the proposal that diphosphonates inhibit IspH through interaction with the active site, thereby preventing HMB-PP from binding to the 4Fe-4S cluster.…”

Biometallorganische Chemie mit IspG und IspH: Struktur, Funktion und Hemmung der an der Isoprenoid‐Biosynthese beteiligten [Fe4S4]‐Proteine

“…Zweitens wurden die vermuteten Radikalspezies in keinem Experiment beobachtet, und die beobachteten Spezies waren keine freien Radikale. Stattdessen fanden wir in quantenchemischen Berechnungen heraus, [59] [21] Diese Struktur ähnelt der von uns früher für die Zwischenstufe X in der IspG-Katalyse (siehe unten) vorgeschlagenen; [31,33,66,67] allerdings stimmt die Beteiligung von Ferraoxetan 5 in der IspH-Reaktion mit mehreren experimentellen Beobachtungen nicht überein; vor allem das Fehlen einer 17 O-Hyperfeinwechselwirkung in Proben, die mit [1-17 O]-4 hergestellt wurden, [50] schließt die Mçglichkeit einer Fe-O-Bindung aus. Zudem gibt es keinen Beweis für eine direkte bindende Wechselwirkung zwischen dem apikalen Fe und C2: die 13 C-Hyperfeinkopplungskonstanten für C1, C2 und C3 sind alle klein (1.8-3.2 MHz; Abbildung 6 b) und kçnnen in DFT-Rechnungen an einem h 3 -Allyl-Modellkomplex reproduziert werden, [59] während die Fe-C2- 13 C-Hyperfeinkopplung im Ferraoxetan 17 MHz beträgt.…”