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...

Posttranscriptional modifications of ribosomal RNA (rRNA) nucleotides are a common mechanism of modulating the ribosome’s function and conferring bacterial resistance to ribosome-targeting antibiotics. One such modification is methylation of an adenosine nucleotide within the peptidyl transferase center of the ribosome mediated by the indigenous methyltransferase RlmN and its evolutionary-related resistance enzyme Cfr. These methyltransferases catalyze methyl transfer to aromatic carbon atoms of the adenosine within a complex 23S rRNA substrate to form the 2,8-dimethylated product. RlmN and Cfr are members of the Radical SAM superfamily, and contain the characteristic cysteine rich CX3CX2C motif. We demonstrate that both enzymes are capable of accommodating the requisite [4Fe-4S] cluster. S-adenosylmethionine (SAM) is both the methyl donor and the source of a 5′-deoxyadenosyl radical, which activates the substrate for methylation. Detailed analyses of the rRNA requirements show that the enzymes can utilize protein-free 23S rRNA as a substrate, but not the fully-assembled large ribosomal subunit, suggesting that the methylations take place during the assembly of the ribosome. The key recognition elements in the 23S rRNA are helices 90–92 and the adjacent single stranded RNA that encompasses A2503. To our knowledge, this study represents the first in vitro description of a methyl transfer catalyzed by a member of Radical SAM superfamily, and it expands the catalytic repertoire of this diverse enzyme class. Furthermore, by providing information on both the timing of methylation and its substrate requirements, our findings have important implications for the functional consequences of Cfr-mediated modification of rRNA in acquisition of antibiotic resistance.

In the malaria parasite Plasmodium falciparum isoprenoid precursors are synthesised inside a plastid-like organelle (apicoplast) by the mevalonate independent 1-deoxy-D D-xylulose-5-phosphate (DOXP) pathway. The last reaction step of the DOXP pathway is catalysed by the LytB enzyme which contains a [4Fe-4S] cluster. In this study, LytB of P. falciparum was shown to be catalytically active in the presence of an NADPH dependent electron transfer system comprising ferredoxin and ferredoxin-NADP + reductase. LytB and ferredoxin were found to form a stable protein complex. These data suggest that the ferredoxin/ferredoxin-NADP + reductase redox system serves as the physiological electron donor for LytB in the apicoplast of P. falciparum.