Cloning and functional characterization of an enzyme from Helicobacter pylori that catalyzes two steps of the methylerythritol phosphate pathway for isoprenoid biosynthesis Jordi Pérez-Gil, María Bergua, Albert Boronat, Santiago Imperial ⁎ Departament de Bioquimica i Biologia Molecular. Universitat de Barcelona, Avda Diagonal 645. 08028-Barcelona, Spain abstract article info Article history: Received 8 December 2009 Received in revised form 15 June 2010 Accepted 16 June 2010 Available online 23 June 2010 Keywords: 4-diphosphocytidyl-2-C-methyl-D-erythritol synthase 2-C-methyl-D-erythritol-2,4-cyclodiphosphate synthase Helicobacter pylori Isoprenoid biosynthesis Methylerythritol phosphate pathway Background: The methylerythritol phosphate pathway for isoprenoid biosynthesis is an attractive target for the design of new specific antibiotics for the treatment of gastrointestinal diseases associated with the presence of the bacterium Helicobacter pylori since this pathway which is essential to the bacterium is absent in humans. Results: This work reports the molecular cloning of one of the genes of the methylerythritol phosphate pathway form H. pylori (ispDF; HP_1440) its expression in Escherichia coli and the functional characterization of the recombinant enzyme. As shown by genetic complementation and in vitro functional assays the product of the ispDF gene form H. pylori is a bifunctional enzyme which can replace both CDP-methylerythritol synthase and methylerythritol cyclodiphosphate synthase from E. coli. General significance: Designing inhibitors that affect at the same time both enzyme activities of the H. pylori bifunctional enzyme (i.e. by disrupting protein oligomerization) would result in more effective antibiotics which would be able to continue their action even if the bacterium acquired a resistance to another antibiotic directed against one of the individual activities. Conclusion: The bifunctional enzyme would be an excellent target for the design of new, selective antibiotics for the treatment of H. pylori associated diseases. © 2010 Elsevier B.V. All rights reserved. 1. Introduction Isoprenoids are the most diverse group of natural products with more than 35,000 compounds identified to date [1,2]. Isoprenoids are synthesized in all living organisms and play essential roles in membrane structure (sterols and hopanoids), redox reactions (ubiquinone, menaquinone, and plastoquinone), light harvesting and photoprotection (carotenoids and side chain of chlorophylls), and regulation of growth and development (steroid hormones, gibberellins, cytokinins, abscisic acid, and substrates for protein modification) [1–3]. All isoprenoids derive from two common C 5 units: isopentenyl diphosphate (IPP) and its isomer dimethylallyl diphosphate (DMAPP). Until the early 1990s, it was accepted that IPP and DMAPP biosynthesis proceeded in all organisms through the same set of enzyme reactions, the mevalonate pathway. However, an alternative pathway for the synthesis of IPP and DMAPP has been identified in eubacteria [3], green algae [4] and plants [5,6], the methylerythritol phosphate (MEP) pathway. The genes and enzymes of the MEP pathway are best characterized in Escherichia coli (Fig. 1). In the initial reaction 1-deoxy-D-xylulose 5-phosphate (DXP) is formed by condensation of D-glyceraldehyde 3-phosphate with (hydroxyethyl) thiamine derived from pyruvate. This reaction is catalyzed by the enzyme DXP synthase (DXS) encoded by the dxs gene [7–9]. In the next step, DXP is converted into 2-C-methyl-D-erythritol 4-phosphate (methylerythritol phosphate or MEP) by intramolecular rearrangement and reduction in a reaction catalyzed by DXP reductoisomerase (DXR) [10–12]. This is the first committed step of the pathway and MEP represents the first specific intermediate. In the third step MEP is converted to the CDP-derivative 4-diphosphocyti- dyl-2-C-methyl-D-erythritol (CDP-ME) by the enzyme CDP-ME synthase (CMS), the protein product of the E. coli gene ygbP (also called ispD) [13–15]. In the following reaction step CDP-ME is phosphorylated in an ATP-dependent reaction by the enzyme CDP- ME kinase (CMK) to yield 4-diphosphocytidyl-2-C-methyl-D-erythri- tol 2-phosphate (CDP-MEP) [16,17]. This enzyme is specified by the ychB (or ispE) gene from E. coli [16]. CDP-MEP is next transformed into CMP and 2-C-methyl-D-erythritol-2,4-cyclodiphosphate (MECDP) by the enzyme MECDP synthase (MCDS) encoded by the ygbB (or ispF) gene [18]. Finally, the gcpE (ispG) protein product, hydroxymethyl- butenyl 4-diphosphate synthase (HMBS) and the enzyme specified by Biochimica et Biophysica Acta 1800 (2010) 919–928 ⁎ Corresponding author. Tel.: + 34 93 4021873; fax: + 34 93 4021559. E-mail address: simperial@ub.edu (S. Imperial). 0304-4165/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.bbagen.2010.06.008 Contents lists available at ScienceDirect Biochimica et Biophysica Acta journal homepage: www.elsevier.com/locate/bbagen