(MEP) Pathway for Isoprenoid Biosynthesis in Bacteria
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A new family of enzymes catalyzing the first committed step of the methylerythritol 4-phosphate (MEP) pathway for isoprenoid biosynthesis in bacteria Félix J. Sangaria,1, Jordi Pérez-Gilb, Lorenzo Carretero-Pauletc,d, Juan M. García-Loboa, and Manuel Rodríguez-Concepciónb,1 aDepartamento de Biología Molecular, Universidad de Cantabria and Instituto de Biomedicina y Biotecnologia de Cantabria (IBBTEC), CSIC-UC-IDICAN, 39011 Santander, Spain; bDepartment of Molecular Genetics, Centre for Research in Agricultural Genomics (CRAG) CSIC-IRTA-UAB, 08034 Barcelona, Spain; cDepartment of Applied Biology (Area of Genetics), University of Almería, 04120 Almería, Spain; and dFaculty of Life Sciences, University of Manchester, Michael Smith Building, Oxford Road, Manchester M13 9PT, United Kingdom Edited* by Elisabeth Gantt, University of Maryland, College Park, MD, and approved May 18, 2010 (received for review February 18, 2010) Isoprenoids are a large family of compounds with essential func- Because the MEP pathway is absent from animals (including tions in all domains of life. Most eubacteria synthesize their isopre- humans) but it is essential in a large number of major bacterial noids using the methylerythritol 4-phosphate (MEP) pathway, pathogens, it has been proposed as a promising new target for the whereas a minority uses the unrelated mevalonate pathway and development of novel antiinfective agents (8, 9). However, infor- only a few have both. Interestingly, Brucella abortus and some mation on possible mechanisms for resistance to a block of the other bacteria that only use the MEP pathway lack deoxyxylulose MEP pathway is scarce. Antibiotic resistance can be caused by an 5-phosphate (DXP) reductoisomerase (DXR), the enzyme catalyzing active export or blocked import of the drug, by its inactivation the NADPH-dependent production of MEP from DXP in the first inside the cell, by the genetic modification of its protein target, committed step of the pathway. Fosmidomycin, a specific compet- or by the use of an alternative pathway not affected by the inhi- itive inhibitor of DXR, inhibited growth of B. abortus cells expres- bitor, to mention just a few possibilities. The best characterized sing the Escherichia coli GlpT transporter (required for fosmidomy- inhibitor of the MEP pathway is fosmidomycin (FSM), first iden- cin uptake), confirming that a DXR-like (DRL) activity exists in these tified as a natural antibiotic effective against a wide bacterial bacteria. The B. abortus DRL protein was found to belong to a fa- spectrum. FSM is a specific competitive inhibitor of deoxyxylu- mily of uncharacterized proteins similar to homoserine dehydro- lose 5-phosphate (DXP) reductoisomerase (DXR), the enzyme genase. Subsequent experiments confirmed that DRL and DXR catalyzing the NADPH-dependent production of MEP from catalyze the same biochemical reaction. DRL homologues shown DXP in the first committed step of the pathway (10). The uptake to complement a DXR-deficient E. coli strain grouped within the of FSM by bacterial cells is an active process involving a cAMP- same phylogenetic clade. The scattered taxonomic distribution dependent glycerol 3-phosphate transporter (GlpT) protein (11). of sequences from the DRL clade and the occurrence of several A defective glpT gene in Escherichia coli mutants or the absence paralogues in some bacterial strains might be the result of lateral of a GlpT homologue in other bacteria such as Mycobacterium gene transfer and lineage-specific gene duplications and/or losses, tuberculosis leads to FSM resistance (11, 12). Overexpression similar to that described for typical mevalonate and MEP pathway of the E. coli fsr gene, encoding a protein with similarity to genes. These results reveal the existence of a novel class of oxidor- bacterial drug-export proteins, also results in FSM resistance, eductases catalyzing the conversion of DXP into MEP in prokaryotic likely because this protein facilitates the export of the inhibitor cells, underscoring the biochemical and genetic plasticity achieved (13). Furthermore, a number of independent mutations have been shown to rescue the survival of E. coli strains defective in by bacteria to synthesize essential compounds such as isoprenoids. BIOCHEMISTRY the first two genes of the MEP pathway, suggesting that bacteria Brucella ∣ DXR ∣ fosmidomycin ∣ methylerythritol ∣ phylogenetic can respond to a block of DXP synthase (DXS) or DXR activities by using other enzymes that produce DXP or MEP when mutated (14). Alternative pathways and metabolic intermediates have soprenoids, one of the largest groups of natural compounds, been proposed to be used for the biosynthesis of isoprenoid pre- have a variety of roles in respiration, photosynthesis, membrane I cursors in the cyanobacterium Synechocystis PCC 6803, which structure, allelochemical interactions, and growth regulation lacks the MVA pathway and does not use the MEP pathway un- (1–3). All free-living organisms synthesize isoprenoids from the der photosynthetic conditions (15, 16). These results illustrate five carbon precursors isopentenyl diphosphate (IPP) and its how limited is still our knowledge of the alternative pathways that double-bond isomer dimethylallyl diphosphate (DMAPP). For can be used by bacteria to synthesize their isoprenoids. In this decades it was believed that IPP was exclusively synthesized from context, we noticed that the completely sequenced genomes of acetyl coenzyme A by the mevalonate (MVA) pathway and then a number of bacteria, including the pathogenic Brucella abortus converted into DMAPP by a IPP/DMAPP isomerase (IDI) en- 2308 (17), contain the genes of the MEP pathway with the only zyme (4). However, in the early nineties of the last century it exception of that encoding DXR, suggesting that these bacteria was discovered that IPP and DMAPP could be formed simulta- neously from pyruvate and glyceraldehyde 3-phosphate by an alternative route currently known as the methylerythritol 4-phos- Author contributions: F.J.S., J.P.-G., and M.R.-C. designed research; F.J.S. and J.P.-G. phate (MEP) pathway (5, 6). It is now well established that most performed research; F.J.S., J.P.-G., L.C.-P., and J.M.G.-L. contributed new reagents/analytic tools; F.J.S., J.P.-G., L.C.-P., J.M.G.-L., and M.R.-C. analyzed data; and F.J.S. and M.R.-C. wrote organisms only use one of the two pathways for isoprenoid bio- the paper. synthesis. Thus, archaea (archaebacteria), fungi, and animals The authors declare no conflict of interest. synthesize IPP from MVA, whereas most bacteria (eubacteria) *This Direct Submission article had a prearranged editor. only use the MEP pathway for the production of isoprenoid pre- 1To whom correspondence may be addressed. E-mail: [email protected] or mrcgmp@cid. cursors. Plants employ both pathways, but in different cell com- csic.es. partments: The MVA pathway synthesizes cytosolic isoprenoid This article contains supporting information online at www.pnas.org/lookup/suppl/ precursors whereas the MEP pathway is located in plastids (7). doi:10.1073/pnas.1001962107/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1001962107 PNAS ∣ August 10, 2010 ∣ vol. 107 ∣ no. 32 ∣ 14081–14086 Downloaded by guest on October 1, 2021 may use an alternative enzyme to produce MEP. Here we report was actually inhibited by FSM, consistent with the hypothesis that the cloning of the gene encoding such an alternative enzyme, the biochemical mechanism used by this alternative enzyme to demonstrate its biochemical activity both in vivo and in vitro, produce MEP from DXP might be similar to that used by DXR. and examined its phylogenetic distribution across bacteria. Complementation of E. coli Strains Defective in DXR Leads to the Iden- Results tification of DRL. To identify the gene encoding DRL in B. abortus, B. abortus Cells Expressing GlpT Become Sensitive to FSM. The com- we constructed a genomic library of this bacterium and used it to pletely sequenced genome of B. abortus lacks a DXR-encoding complement a DXR-deficient E. coli mutant. The genome of gene but it contains homologues of the rest of MEP pathway E. coli strain EcAB4-10 harbours a deletion of the dxr gene enzymes (Fig. 1 and Table S1). This suggests that a B. abortus and a synthetic MVA operon that allows the production of protein showing no overall homology to DXR might be involved IPP and DMAPP (and therefore the survival of the cells) when in transforming DXP into MEP. However, growth of B. abortus MVA is supplied to the growth medium (14). Competent EcAB4- cells was not inhibited by concentrations of FSM up to 1 mg∕ 10 cells were transformed with the B. abortus genomic library and mL. This result suggested that the putative DXR-like (DRL) en- plated in the absence of MVA. Plasmids from positive transfor- zyme might not be inhibited by FSM or, alternatively, that the mants that grew without exogenous MVA were sequenced and inhibitor was degraded, expelled, or not taken by living cells. Con- shown to contain overlapping B. abortus genomic fragments con- sistent with the last possibility, the B. abortus genome contains a taining the genes BAB2_0264 and BAB2_0265 (Fig. S2A). fsr gene (BAB1_0676) but no glpT homologue. To investigate BAB2_0265 codes for a hydrolase whereas BAB2_0264 codes whether a defective uptake of FSM was the cause of the resis- for a protein annotated as a putative oxidoreductase (a family tance phenotype of this bacterium, the E. coli gene encoding that includes DXR) and was therefore selected for further experi- the GlpT transporter was expressed in B. abortus cells. As shown ments. Transformation of EcAB4-10 cells with a vector harbour- in Fig. S1, transformants became FSM sensitive with a minimal ing only BAB2_0264 led to full complementation of MVA inhibitory concentration of 4 μg∕mL, confirming that the resis- auxotrophy (Fig. 2), suggesting that the encoded protein tance phenotype of wild-type cells resulted solely from the (Q2YIM3) was the predicted B.