In all organisms studied so far, isoprenoids such as dolichol, ubiquinones, carotenoids, and sterols are synthesized from isopentenyl diphosphate (IPP) and its isomer, dimethylallyl diphosphate (DMAPP). However, there are two completely different biosynthetic pathways leading to these two precursor molecules. In animals, fungi, archaea, and some bacteria, IPP and DMAPP are synthesized via the well-known mevalonate pathway. In contrast, the vast majority of bacteria, and some parasitic protozoa of the phylum Apicomplexa, synthesize IPP and DMAPP via the 2-C-methyl-D-erythritol-4-phosphate (MEP) pathway [also known as the 1-deoxy-D-xylulose-5-phosphate (DOXP), or non-mevalonate pathway]. [1][2][3] Since the MEP pathway is not used by humans, it represents an attractive target for the development of new antimicrobial compounds and indeed, inhibitors of the second enzyme of the MEP pathway, DOXP reductoisomerase, have demonstrated good antibacterial as well as antimalarial activity, in clinical settings. [4][5][6] Both IPP and DMAPP are formed (in a ~5:1 ratio) in the last step of the MEP pathway from the substrate (E)-4-hydroxy-3-methyl-but-2-enyl diphosphate (HMBPP) in the following reaction: catalyzed by the enzyme HMBPP reductase, also known as LytB or IspH. 7 The reaction is thought to involve an iron-sulfur cluster reducing HMBPP to an allylic anion, followed by protonation at either C2 or C4, to form IPP or DMAPP. 8 However, the three-dimensional structure of the LytB enzyme has not yet been reported, making mechanistic analyses more challenging. Here, we report the X-ray crystallographic structure of the LytB enzyme from Aquifex aeolicus and propose a structure-based model for catalysis.We first discuss several features of LytB sequences in general that might be expected to be of importance for substrate binding and catalysis by considering the sequence homology between 224 LytB enzymes as annotated by the JPRED3 program. 9 There are three totally conserved cysteines in the A. aeolicus sequence that anchor the catalytically active iron-sulfur cluster: Cys13, Cys96, and Cys193 (Figure 1 and Supporting Information, Figure S1). HMBPP can be expected to bind to the iron-sulfur cluster during catalysis via its O4 atom, but it also needs to bind to protein residues and related diphosphates typically bind to prenyl synthase enzymes either via a DDXXD motif or via electrostatic/hydrogen bond interactions with Lys, Arg, or His. On the basis of the sequence alignment (Supporting Information Figure S1) we find neither evidence for DDXXD clusters, consistent with the lack of a requirement for Mg 2+ , nor totally conserved Lys or Arg residues or even totally conserved Lys/Arg positions. There are, however, two highly conserved His residues, H42 and H124. These two conserved His were reported in 10 putative LytB sequences by Adam et al. 11 and are now seen in all 224 sequences. This strongly suggests that the diphosphate binds to H42 and H124. The third feature that would be expected for LytB is the presence of a totally...

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)

a b s t r a c tTerpenoid precursor biosynthesis occurs in human and many pathogenic organisms via the mevalonate and 2-C-methyl-D-erythritol-4-phosphate (MEP) pathways, respectively. We determined the X-ray structure of the Fe/S containing (E)-4-hydroxy-3-methyl-but-2-enyl-diphosphate reductase (LytB) of the pathogenic protozoa Plasmodium falciparum which catalyzes the terminal step of the MEP pathway. The cloverleaf fold and the active site of P. falciparum LytB corresponds to those of the Aquifex aeolicus and Escherichia coli enzymes. Its distinct electron donor [2Fe-2S] ferredoxin was modeled to its binding site by docking calculations. The presented structural data provide a platform for a rational search of anti-malarian drugs.

Edited by Stuart Ferguson Keywords:Isoprenoid biosynthesis GcpE Iron-sulfur cluster X-ray structure Drug design a b s t r a c t Isoprenoid precursor biosynthesis occurs through the mevalonate or the methylerythritol phosphate (MEP) pathway, used i.e., by humans and by many human pathogens, respectively. In the MEP pathway, 2-C-methyl-D-erythritol-2,4-cyclo-diphosphate (MEcPP) is converted to (E)-1-hydroxy-2-methyl-but-2-enyl-4-diphosphate (HMBPP) by the iron-sulfur cluster enzyme HMBPP synthase (GcpE). The presented X-ray structure of the GcpE-MEcPP complex from Thermus thermophilus at 1.55 Å resolution provides valuable information about the catalytic mechanism and for rational inhibitor design. MEcPP binding inside the TIM-barrel funnel induces a 60°rotation of the [4Fe-4S] cluster containing domain onto the TIM-barrel entrance. The apical iron of the [4Fe-4S] cluster ligates with the C3 oxygen atom of MEcPP.

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