The alkyne group of 11 is shifted compared with 5, thus there is no interaction with the iron and the carbon chain is rotated towards a hydrophobic pocket formed by A73 and H74

The alkyne group of 11 is shifted compared with 5, thus there is no interaction with the iron and the carbon chain is rotated towards a hydrophobic pocket formed by A73 and H74. IspH. Introduction Metalloproteins containing [Fe4S4] clusters carry out a broad series of reactions1,2. For example, aconitase3 catalyses the hydration of the alkene-containing species (Fig. 2). All compounds gave similar M??bauer spectra (Fig. 3aCc) with quadrupole splittings (EQ) of ~1.1 mm s?1 and isomer shifts (Fe) of ~0.45 mm s?1. These values differ from those seen in ligand-free IspH (Fig. 3d)19. However, they are similar to those observed on addition of the HMBPP substrate (1) to the enzyme, where the quadrupole splitting of the fourth Fe changes from 1.91 to 1 1.00 mm s?1, and the isomer shifts decrease from 0.89 to 0.53 mm s?119. As noted by Seemann IspH bound to: (a) propynyl diphosphate 4 (Component 1 (25%) =0.52 mm s?1, EQ=1.54 mm s?1; Component 2 (50%)=0.45 mm s?1, EQ=1.20 mm s?1; Component 3 (25%) =0.41 mm s?1, EQ=0.85 mm s?1); (b) but-3-ynyl diphosphate 5 (Component 1 (50%) =0.45 mm s?1, Tolfenamic acid EQ=1.28 mm s?1; Component 2 (50%) =0.39 mm s?1, EQ=0.89 mm s?1); (c) pent-4-ynyl diphosphate 6 (Component 1 (50%) =0.42 mm s?1, EQ=1.54 mm s?1; Component 2 (50%) =0.45 mm s?1, EQ=1.20 mm s?1); (d) ligand-free IspH (Component 1 (50%) =0.43 mm s?1, EQ=1.28 mm s?1; Component 2 (25%)=0.38 mm s?1, EQ=0.86 mm s?1; Component 3 (25%) =0.82 mm s?1, EQ=1.98 mm s?1). Propynyl diphosphate (4) is not converted by Tolfenamic acid IspH Subsequently, we performed co-crystallization of compounds 4C6 bound to oxidised IspH and analysed the ligand-bound X-ray structures obtained. As can be seen with 4 (Fig. 4a), the acetylene group is not bonded to the fourth Fe (FeCC distances of 3.4C3.5 ?), rather, there is a water molecule (or, in principle, a hydroxide ion) bound to the [Fe4S4] cluster with a Fe-O bond length of 2.1 ?, essentially the same as in the alkoxide complex formed by oxidised IspH and HMBPP (1)21. The crystal structure of IspH in complex with 4 gives an explanation of the 57Fe M??bauer results: the Fe and EQ values for 1 as well as 4 bound to IspH are essentially the same because both species contain a [Fe4S4] cluster with 3S,1O coordination to the apical Fe. Open in a separate Tolfenamic acid Tolfenamic acid window Figure 4 Stereo presentation of the IspH active site with ligands 4 and 7Ligands, [Fe4S4] clusters and side chains of E126 as well as T167 are displayed as stick models. The protein backbone is presented as coil model (grey) and solvent molecules as spheres (red). A omit electron density map (blue mesh, contoured at 1.4 ) is shown for the [Fe4S4] cluster, the ligand, and the solvent molecules in the first coordination sphere; dotted lines indicate coordination and hydrogen bonds. (a) Crystal structure of IspH bound to propynyl diphosphate (4). The diphosphate group of the ligand binds in the same orientation as observed for the substrate Tolfenamic acid 1 (ref. 21), but no interaction between the alkyne group and the ironCsulphur cluster were observed. Two water molecules are located in the central cavity: W3 coordinates to the fourth site of the Rabbit Polyclonal to IKK-gamma (phospho-Ser376) apical iron of the [Fe4S4] cluster and W2 is stabilized by E126O as well as the diphosphate group. (b) IspH active site with the 1-enolate 7 and water molecule W1. IspH hydrates but-3-ynyl diphosphate (5) to the corresponding aldehyde 8. IspH converts but-3-ynyl diphosphate (5) to an aldehyde Although the M??bauer spectroscopic results with 4 and 5 are similar, the X-ray structure of IspH in complex with 5 reveals, surprisingly, an enolate complex (Fig. 4b). The diphosphate backbone of 5 binds to the same pocket.