Structure of theE-1-hydroxy-2-methyl-but-2-enyl-4-diphosphate synthase (GcpE) fromThermus thermophilus

Abstract

Edited by Richard CogdellKeywords: Non-mevalonate pathway E-1-hydroxy-2-methyl-but-2-enyl-4-diphosphate synthase Iron-sulfur cluster X-ray structure Drug design a b s t r a c t Isoprenoids are biosynthesized via the mevalonate or the 2-C-methyl-D-erythritol-4-phosphate (MEP) pathways the latter being used by most pathogenic bacteria, some parasitic protozoa, plant plastids, but not by animals. We determined the X-ray structure of the homodimeric [4Fe-4S] cluster carrying E-1-hydroxy-2-methyl-but-2-enyl-4-diphosphate synthase (GcpE) of Thermus thermophilus which catalyzes the penultimate reaction of the MEP pathway and is therefore an attractive target for drug development. The [4Fe-4S] cluster ligated to three cysteines and one glutamate is encapsulated at the intersubunit interface. The substrate binding site lies in front of an (ab) 8 barrel. The great [4Fe-4S] cluster-substrate distance implicates large-scale domain rearrangements during the reaction cycle.Structured summary: gcpE binds to gcpE by x-ray crystallography (View interaction)

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

“…To provide possible answers to these questions, we first used several different protein structure prediction programs (27)(28)(29)(30)(31) to make predictions of the structure of the insert domain A*. Remarkably, in all cases, the structure predictions for the A* insert domain converged to the known N-terminal TIM barrel fold (A) found in the two bacterial IspG structures (7,9). A representative structure (using a ClustalW2 multisequence alignment and Modeller) (32,33) is shown in Fig.…”

“…If, on the other hand, the three-domain catalytically-active structures are monomeric, then a structural organization similar to that shown in Fig. 1H could be envisaged, in which the insert domain would play a similar "structural" role to the TIM barrels in the bacterial IspGs in which there is a large A/A contact patch in the ðABÞ 2 dimer interface (7,9) that stabilizes the protein's structure. The AA* domain would thus act as a rigid rod with the B domain moving onto the rod to effect reduction of the reaction intermediate, then off of it to permit product release: a "cup-and ball" model.…”

“…1D, a ConSurf (10) analysis reveals many highly conserved residues (dark pink) in the TIM barrel and at the A/B interface in the dimer. IspG could, however, also in principle function as a monomer (or higher multimer), although the dimer model is more satisfying because the distance between the center of the TIM barrel is only approximately 15 Å from the 4Fe4S cluster (in AaIspG; approximately 20 Å in TtIspG) as compared with an approximately 50 Å distance to the TIM barrel in the same chain, and can be "bent" to be even closer (9), as seen in Fig. 1 E and F. These observations then raise two key questions: First, is the catalytic dimer model in fact correct?…”

“…In recent work, X-ray crystallographic structures of IspG from Aquifex aeolicus and Thermus thermophilus (called here AaIspG and TtIspG) have been reported (7,9) and these provide important clues as to the likely nature of the 3-domain proteins. The structure of the (A,B) 2-domain protein AaIspG is illustrated in Fig.…”

“…1C and consists of a dimer of IspG molecules. There are two domains in each monomer, with the N terminus (A, blue) having close structural homology with the TIM (triose phosphate isomerase) barrel in dihydropteroate synthase, and the C terminus (B, orange) having close structural homology to sulfite/nitrite reductase (7,9). Based on these results, it was proposed that the dimer ðABÞ 2 is the catalytically relevant structure, with the C terminus (4Fe4S cluster, B) of one molecule in the dimer interacting with the open mouth of the TIM barrel (A) in the second molecule in the dimer in such a way that the MEcPP substrate is trapped between the two domains, interacting with both.…”