Structure of Active IspH Enzyme from Escherichia coli Provides Mechanistic Insights into Substrate Reduction

Abstract

Eukaryotes and most prokaryotes require isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) as biosynthetic precursors of terpenes. Whereas animals generate these essential metabolites via the mevalonate pathway, [1] many human pathogens including Plasmodium falciparum and Mycobacterium tuberculosis are known to use the more recently identified non-mevalonate pathway, which is a potential target for drug development.[2-4] The final step of this pathway is catalyzed by IspH protein, which generates a mixture of IPP and DMAPP by reductive dehydration of 1-hydroxy-2-methyl-2-(E)-butenyl-4-diphosphate (HMBPP, Figure 1 a). [5][6][7][8][9][10][11] Recently, Rekittke et al. described the first X-ray structure of IspH protein from the hyperthermophilic eubacterium Aquifex aeolicus in its open state.[12] Herein, we report the crystal structure of the IspH protein from Escherichia coli [11] in its closed conformation, which serves as basis for a detailed discussion of the catalytic pathway.Recombinant E. coli IspH protein (comprising an Nterminal His 6 fusion tag) was purified and crystallized under anaerobic conditions. Its structure was determined to a resolution of 1.8 by single-wavelength anomalous diffraction methods. Three iron sites per protein unit were localized in the anomalous difference Patterson map and were used for phasing. Successive rounds of model building and refinement afforded a well-defined electron density for the entire IspH molecule except for the N-terminal His 6 tag and five Cterminal amino acid residues (R free = 23.8 %, Supporting Information, Table S2). The root mean square (r.m.s.) deviation between the C a positions of the two protein molecules in the asymmetric unit is less than 0.3 . The folding pattern of the monomeric protein involves three structurally similar domains, D1 to D3, which are related by pseudo-C 3 symmetry but are devoid of detectable sequence similarity (Figure 1 b and Supporting Information, Figure S1). Relative to domain D1, domains D2 and D3 appear rotated by angles of approximately 1008 and 1408, respectively. Each domain starts with a conserved cysteine residue that protrudes into a cavity at the center of the protein where it coordinates one respective iron atom of a [3Fe-4S] cluster. The cluster appears to be tilted relative to the pseudotrigonal axis of the apoprotein by about 208. The trigonal symmetric [3Fe-4S] cluster is located in a hydrophobic pocket of the central cavity, which is formed by residues located on D1 (G14 and V15), D2 (P97 and V99), D3 (A199) as well as the C-terminus (F302 and P305), which stabilizes the arrangement of the individual domains. Furthermore, the methylene moiety of C96 in D2 is turned inward generating additional hydrophobic shielding of atom Fe 2 (see Figure 2). Residual electron density located inside the central cavity was identified as inorganic diphosphate (PP i ; see Supporting

Probing the reaction mechanism of IspH protein by x-ray structure analysis

“…Interestingly, it only was possible to collect a full dataset of the intact HMBPP ligand bound to IspH when exposed to a significantly short irradiation time (4 min). The crystal structure of the complex reveals a closed conformation of IspH with the position of the protein backbone being virtually identical to that previously reported for E. coli IspH protein in complex with inorganic pyrophosphate (rmsd, 0.3 Å) (5). Inside the central cavity, the electron density of the bound substrate is well defined.…”

“…The pyrophosphate moiety assumes the same position as the inorganic pyrophosphate ion and is embedded in a polar environment comprising the side chains of histidines 41, 74, and 124, serines 225 and 269, threonine 168, asparagine 227 and glutamine 166. The overall conformation of the bound substrate is hairpin-shaped resembling the recently published modeled ligand (5). A single fixed water molecule (subsequently designated W1) is well-defined inside the densely packed active site cavity where it is hydrogen bonded to Author contributions: T.G., W.E., F.R., A.B., and M.G.…”

“…At its center of mass, the protein features an iron-sulfur cluster which is coordinated by the cysteine residues 12, 96, and 197 (one from each of the three folding domains, amino acid residue numbers refer to E. coli). Although it is mostly assumed that IspH protein requires a [4Fe-4S] cluster for catalytic activity (7,8), all published structures so far showed [3Fe-4S] clusters (5,6). Notably, the E. coli protein crystallized in a closed conformation with the iron-sulfur cluster located inside a solvent-inaccessible central cavity that also contained a pyrophosphate or malonate ion, depending on crystallization conditions (5).…”

“…Ever since the discovery of IspH protein, its reaction mechanism has been the subject of intense investigation (5,9,10). It is generally agreed that at least one intermediate must be an allyl derivative (Fig.…”

Bioorganometallic mechanism of action, and inhibition, of IspH

“…Ligand-free IspH has an "open" structure (12), whereas IspH cocrystallized with diphosphate has a "closed" structure (13) in which a serine-X-asparagine (SXN) loop is involved in hydrogen bonding with a PPi ligand. The mechanism of action of IspH is controversial and there have been many different proposals (13,15,(18)(19)(20)(21) (Fig. S1).…”