Organometallic mechanism of action and inhibition of the 4Fe-4S isoprenoid biosynthesis protein GcpE (IspG)

• Published in: Proceedings of the National Academy of Sciences - PNAS 2010-06, Vol.107 (25), p.11189-11193
• Main Authors: Wang, Weixue, Li, Jikun, Wang, Ke, Huang, Cancan, Zhang, Yong, Oldfield, Eric, Walsh, Christopher T.
• Format: Article
• Language: English
• Subjects: Alkenes; Alkynes; Arabidopsis - metabolism; Arabidopsis Proteins - genetics; Arabidopsis Proteins - physiology; Biochemistry; Biosynthesis; Catalysis; Diphosphates; Drug Design; Electron Spin Resonance Spectroscopy; Enzymes; Epoxy compounds; Iron - chemistry; Kinetics; Metals; Models, Chemical; Organometallic Compounds - chemistry; Oxidoreductases; Phosphates; Physical Sciences; Plant Extracts - pharmacology; Protein Conformation; Proteins; Quadrants; Spectroscopy; Sulfur - chemistry; Terpenes - chemistry; Terpenoids; Thermus thermophilus - metabolism; X-Rays
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.1000264107

We report the results of a series of chemical, EPR, ENDOR, and HYSCORE spectroscopic investigations of the mechanism of action (and inhibition) of GcpE, E-1-hydroxy-2-methyl-but-2-enyl-4-diphosphate (HMBPP) synthase, also known as IspG, an Fe₄S₄ cluster-containing protein. We find that the epoxide of HMBPP when reduced by GcpE generates the same transient EPR species as observed on addition of the substrate, 2-C-methyl-D-erythritol-2, 4-cyclo-diphosphate. ENDOR and HYSCORE spectra of these transient species (using ²H, ¹³C and ¹⁷O labeled samples) indicate formation of an Fe-C-H containing organometallic intermediate, most likely a ferraoxetane. This is then rapidly reduced to a ferracyclopropane in which the HMBPP product forms an η²-alkenyl π- (or π/σ) complex with the 4th Fe in the Fe₄S₄ cluster, and a similar "metallacycle" also forms between isopentenyl diphosphate (IPP) and GcpE. Based on this metallacycle concept, we show that an alkyne (propargyl) diphosphate is a good (K i ∼ 300 nM) GcpE inhibitor, and supported again by EPR and ENDOR results (a ¹³C hyperfine coupling of ∼7 MHz), as well as literature precedent, we propose that the alkyne forms another π/σ metallacycle, an η²-alkynyl, or ferracyclopropene. Overall, the results are of broad general interest because they provide new mechanistic insights into GcpE catalysis and inhibition, with organometallic bond formation playing, in both cases, a key role.