A new family of enzymes catalyzing the first committed step of the methylerythritol 4-phosphate (MEP) pathway for isoprenoid biosynthesis in bacteria

Abstract

Isoprenoids are a large family of compounds with essential functions in all domains of life. Most eubacteria synthesize their isoprenoids using the methylerythritol 4-phosphate (MEP) pathway, whereas a minority uses the unrelated mevalonate pathway and only a few have both. Interestingly, Brucella abortus and some other bacteria that only use the MEP pathway lack deoxyxylulose 5-phosphate (DXP) reductoisomerase (DXR), the enzyme catalyzing the NADPH-dependent production of MEP from DXP in the first committed step of the pathway. Fosmidomycin, a specific competitive inhibitor of DXR, inhibited growth of B. abortus cells expressing the Escherichia coli GlpT transporter (required for fosmidomycin uptake), confirming that a DXR-like (DRL) activity exists in these bacteria. The B. abortus DRL protein was found to belong to a family of uncharacterized proteins similar to homoserine dehydrogenase. Subsequent experiments confirmed that DRL and DXR catalyze the same biochemical reaction. DRL homologues shown to complement a DXR-deficient E. coli strain grouped within the same phylogenetic clade. The scattered taxonomic distribution of sequences from the DRL clade and the occurrence of several paralogues in some bacterial strains might be the result of lateral gene transfer and lineage-specific gene duplications and/or losses, similar to that described for typical mevalonate and MEP pathway genes. These results reveal the existence of a novel class of oxidoreductases catalyzing the conversion of DXP into MEP in prokaryotic cells, underscoring the biochemical and genetic plasticity achieved by bacteria to synthesize essential compounds such as isoprenoids.I soprenoids, one of the largest groups of natural compounds, have a variety of roles in respiration, photosynthesis, membrane structure, allelochemical interactions, and growth regulation (1-3). All free-living organisms synthesize isoprenoids from the five carbon precursors isopentenyl diphosphate (IPP) and its double-bond isomer dimethylallyl diphosphate (DMAPP). For decades it was believed that IPP was exclusively synthesized from acetyl coenzyme A by the mevalonate (MVA) pathway and then converted into DMAPP by a IPP/DMAPP isomerase (IDI) enzyme (4). However, in the early nineties of the last century it was discovered that IPP and DMAPP could be formed simultaneously from pyruvate and glyceraldehyde 3-phosphate by an alternative route currently known as the methylerythritol 4-phosphate (MEP) pathway (5, 6). It is now well established that most organisms only use one of the two pathways for isoprenoid biosynthesis. Thus, archaea (archaebacteria), fungi, and animals synthesize IPP from MVA, whereas most bacteria (eubacteria) only use the MEP pathway for the production of isoprenoid precursors. Plants employ both pathways, but in different cell compartments: The MVA pathway synthesizes cytosolic isoprenoid precursors whereas the MEP pathway is located in plastids (7).Because the MEP pathway is absent from animals (including humans) but it is essential in a large number ...

Crystal Structure of Brucella abortus Deoxyxylulose-5-phosphate Reductoisomerase-like (DRL) Enzyme Involved in Isoprenoid Biosynthesis

“…The SeMet-labeled protein was produced by the methionine pathway inhibition method (29). PCR amplification of the pET-DRL plasmid (25) with complementary primers containing the desired mutations (E209S/R213A-F, 5Ј-GATTACAGGCA-ATCAGCAGACCGCGCGAACATGAATGTG; and E209S/ R213A-R, 5Ј-CACATTCATGTTCGCGCGGTCTGCTGAT-TGCCTGTAATC) followed by DpnI digestion generated the construct for the expression of the double-mutant BaDRL protein. Both SeMet-labeled and mutant BaDRL proteins were expressed and purified following the protocol used for the unlabeled wild-type protein.…”

“…Protein Expression and Purification-Protein expression and purification were carried out as described (25). Protein-containing fractions were pooled, concentrated, and loaded onto a HiLoad Superdex 200 16/60 gel filtration column (GE Healthcare) that was equilibrated in buffer solution containing 0.02 M Tris-HCl (pH 7.5), 0.1 M NaCl, 2 mM MgCl 2 , 2 mM DTT, and 5% (v/v) glycerol.…”

“…After incubation at 37°C, complementation was assessed by visually monitoring the development of colonies on plates and by quantifying the growth of liquid cultures by absorbance at 600 nm. In vitro activity was estimated using a photometric assay as described (25). For calculation of IC 50 values, seven concentrations of the inhibitors in the range of 0.016 -100 M were added to the reaction mixtures with 35 g of recombinant BaDRL.…”

“…Thus, two types of isopentenyl diphosphate:dimethylallyl diphosphate isomerase enzymes with no sequence, structural, or catalytic similarity have been found in bacteria (22)(23)(24). In the case of DXR, we recently identified a DXR-like (DRL) enzyme that shows some sequence similarity to DXR only at the level of the NADPH-binding domain but catalyzes the same biochemical reaction (25). DRL is found instead of DXR in emerging animal and human pathogens such as Bartonella (which is responsible for a variety of HIV-associated infections) and Brucella (which usually infects livestock but can also spread to humans) (26 -28).…”

“…DRL is found instead of DXR in emerging animal and human pathogens such as Bartonella (which is responsible for a variety of HIV-associated infections) and Brucella (which usually infects livestock but can also spread to humans) (26 -28). Although DRL can be inhibited with FSM (25), it would be most interesting to develop specific antibiotics against DRLharboring pathogens that would not affect beneficial bacteria that use DXR (like those present in the intestine ). However, the identification and development of such narrow-spectrum drugs require structural information for both enzymes.…”