Genetic evidence of branching in the isoprenoid pathway for the production of isopentenyl diphosphate and dimethylallyl diphosphate in Escherichia coli

An alternative mevalonate-independent pathway for isoprenoid biosynthesis has been recently discovered in eubacteria (including Escherichia coli) and plant plastids, although it is not fully elucidated yet. In this work, E. coli cells were engineered to utilize exogenously provided mevalonate and used to demonstrate by a genetic approach that branching of the endogenous pathway results in separate synthesis of the isoprenoid building units isopentenyl diphosphate (IPP) and its isomer dimethylallyl diphosphate (DMAPP). In addition, the IPP isomerase encoded by the idi gene was shown to be functional in vivo and to represent the only possibility for interconverting IPP and DMAPP in this bacterium.     

LytB, a novel gene of the 2-C-methyl-D-erythritol 4-phosphate pathway of isoprenoid biosynthesis in Escherichia coli.

The mevalonate-independent 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway for isoprenoid biosynthesis is essential in many eubacteria, plants, and the malaria parasite. Using genetically engineered Escherichia coli cells able to utilize exogenously provided mevalonate for isoprenoid biosynthesis by the mevalonate pathway we demonstrate that the lytB gene is involved in the trunk line of the MEP pathway. Cells deleted for the essential lytB gene were viable only if the medium was supplemented with mevalonate or the cells were complemented with an episomal copy of lytB.     

fldA is an essential gene required in the 2-C-methyl-D-erythritol 4-phosphate pathway for isoprenoid biosynthesis

Although flavodoxin I is indispensable for Escherichia coli growth, the exact pathway(s) where flavodoxin I is essential has not been identified. We performed transposon mutagenesis of the flavodoxin I gene, fldA, in an E. coli strain that expressed mevalonate pathway enzymes and that had a point mutation in the lytB gene of the MEP pathway resulting in the accumulation of (E)-4-hydroxy-3-methylbutyl-2-enyl pyrophosphate (HMBPP). Disruption of fldA abrogated mevalonate-independent growth and dramatically decreased HMBPP levels. The fldA- mutant grew with mevalonate indicating that the essential role of flavodoxin I under aerobic conditions is in the MEP pathway. Growth was restored by fldA complementation. Since GcpE (which synthesizes HMBPP) and LytB are iron-sulfur enzymes that require a reducing system for their activity, we propose that flavodoxin is essential for GcpE and possibly LytB activity. Thus, the essential role for flavodoxin I in E. coli is in the MEP pathway for isoprenoid biosynthesis.     

Structure and mechanism of 2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase. An enzyme in the mevalonate-independent isoprenoid biosynthetic pathway.

The enzyme 2-C-methyl-D-erythritol 2,4-cyclodiphosphate (MECDP) synthase catalyzes the conversion of 4-diphosphocytidyl-2-C-methyl-D-erythritol 2-phosphate (CDP-ME2P) to MECDP, a highly unusual cyclodiphosphate-containing intermediate on the mevalonate-independent pathway to isopentenyl diphosphate and dimethylallyl diphosphate. We now report two x-ray crystal structures of MECDP synthase refined to 2.8-A resolution. The first structure contains a bound Mn(2+) cation, and the second structure contains CMP, MECDP, and Mn(2+). The protein adopts a homotrimeric quaternary structure built around a central hydrophobic cavity and three externally facing active sites. Each of these active sites is located between two adjacent monomers. A tetrahedrally arranged transition metal binding site, potentially occupied by Mn(2+), sits at the base of the active site cleft. A phosphate oxygen of MECDP and the side chains of Asp(8), His(10), and His(42) occupy the metal ion coordination sphere. These structures reveal for the first time the structural determinants underlying substrate, product, and Mn(2+) recognition and the likely catalytic mechanism accompanying the biosynthesis of the cyclodiphosphate-containing isoprenoid precursor, MECDP.     

Crystal structure of 4-(cytidine 5'-diphospho)-2-C-methyl-D-erythritol kinase,an enzyme in the non-mevalonate pathway of isoprenoid synthesis

The crystal structure of the enzyme 4-(cytidine 5'-diphospho)-2-C-methyl-D-erythritol (CDP-ME) kinase from the thermophilic bacterium Thermus thermophilus HB8 has been determined at 1.7-A resolution. This enzyme catalyzes phosphorylation of the 2-hydroxyl group of CDP-ME, the fourth step of the non-mevalonate pathway, which is essential for isoprenoid biosynthesis in several pathogenic microorganisms. Since this pathway is absent in humans, it is an important target for the development of novel antimicrobial compounds. The structure of the enzyme is similar to the structures of mevalonate kinase and homoserine kinase, members of the GHMP superfamily. Lys8 and Asp125 are active site residues in mevalonate kinase that also appear to play a catalytic role in CDP-ME kinase. Both the mevalonate and the non-mevalonate pathways therefore involve closely related kinases with similar mechanisms. Assaying the enzyme showed that CDP-ME kinase will phosphorylate CDP-ME but not 4-(uridine 5'-diphospho)-2-C-methyl-D-erythritol, indicating the substrate pyrimidine moiety is involved in important interactions with the enzyme.     

A second target of the antimalarial and antibacterial agent fosmidomycin revealed by cellular metabolic profiling

Antimicrobial drug resistance is an urgent problem in the control and treatment of many of the world's most serious infections, including Plasmodium falciparum malaria, tuberculosis, and healthcare-associated infections with Gram-negative bacteria. Because the non-mevalonate pathway of isoprenoid biosynthesis is essential in eubacteria and P. falciparum and this pathway is not present in humans, there is great interest in targeting the enzymes of non-mevalonate metabolism for antibacterial and antiparasitic drug development. Fosmidomycin is a broad-spectrum antimicrobial agent currently in clinical trials of combination therapies for the treatment of malaria. In vitro, fosmidomycin is known to inhibit the deoxyxylulose phosphate reductoisomerase (DXR) enzyme of isoprenoid biosynthesis from multiple pathogenic organisms. To define the in vivo metabolic response to fosmidomycin, we developed a novel mass spectrometry method to quantitate six metabolites of non-mevalonate isoprenoid metabolism from complex biological samples. Using this technique, we validate that the biological effects of fosmidomycin are mediated through blockade of de novo isoprenoid biosynthesis in both P. falciparum malaria parasites and Escherichia coli bacteria: in both organisms, metabolic profiling demonstrated a block of isoprenoid metabolism following fosmidomycin treatment, and growth inhibition due to fosmidomycin was rescued by media supplemented with isoprenoid metabolites. Isoprenoid metabolism proceeded through DXR even in the presence of fosmidomycin but was inhibited at the level of the downstream enzyme, methylerythritol phosphate cytidyltransferase (IspD). Overexpression of IspD in E. coli conferred fosmidomycin resistance, and fosmidomycin was found to inhibit IspD in vitro. This work has validated fosmidomycin as a biological reagent for blocking non-mevalonate isoprenoid metabolism and suggests a second in vivo target for fosmidomycin within isoprenoid biosynthesis, in two evolutionarily diverse pathogens.     

Identification of novel small molecule inhibitors of 4-diphosphocytidyl-2-C-methyl-D-erythritol (CDP-ME) kinase of Gram-negative bacteria

The biosyntheses of isoprenoids is essential for the survival in all living organisms, and requires one of the two biochemical pathways: (a) Mevalonate (MVA) Pathway or (b) Methylerythritol Phosphate (MEP) Pathway. The latter pathway, which is used by all Gram-negative bacteria, some Gram-positive bacteria and a few apicomplexan protozoa, provides an attractive target for the development of new antimicrobials because of its absence in humans. In this report, we describe two different approaches that we used to identify novel small molecule inhibitors of Escherichia coli and Yersinia pestis 4-diphosphocytidyl-2-C-methyl D-erythritol (CDP-ME) kinases, key enzymes of the MEP pathway encoded by the E. coli ispE and Y. pestisipk genes, respectively. In the first approach, we explored existing inhibitors of the GHMP kinases while in the second approach; we performed computational high-throughput screening of compound libraries by targeting the CDP-ME binding site of the two bacterial enzymes. From the first approach, we identified two compounds with 6-(benzylthio)-2-(2-hydroxyphenyl)-4-oxo-3,4-dihydro-2H-1,3-thiazine-5-carbonitrile and (Z)-3-methyl-4-((5-phenylfuran-2-yl)methylene)isoxazol-5(4H)-one scaffolds which inhibited E. coli CDP-ME kinase in vitro. We then performed substructure search and docking experiments based on these two scaffolds and identified twenty three analogs for structure-activity relationship (SAR) studies. Three new compounds from the isoxazol-5(4H)-one series have shown inhibitory activities against E. coli and Y. pestis CDP-ME kinases with the IC(50) values ranging from 7 to 13 μM. The second approach by computational high-throughput screening (HTS) of two million drug-like compounds yielded two compounds with benzenesulfonamide and acetamide moieties which, at a concentration of 20 μM, inhibited 80% and 65%, respectively, of control CDP-ME kinase activity.     

Biosynthesis of isoprenoids: characterization of a functionally active recombinant 2-C-methyl-D-erythritol 4-phosphate cytidyltransferase (IspD) from Mycobacterium tuberculosis H37Rv

Tuberculosis, caused by Mycobacterium tuberculosis, continues to be one of the leading infectious diseases to humans. It is urgent to discover novel drug targets for the development of antitubercular agents. The 2-C-methyl-D-erythritol-4-phosphate (MEP) pathway for isoprenoid biosynthesis has been considered as an attractive target for the discovery of novel antibiotics for its essentiality in bacteria and absence in mammals. MEP cytidyltransferase (IspD), the third-step enzyme of the pathway, catalyzes MEP and CTP to form 4-diphosphocytidyl-2-C-methylerythritol (CDP-ME) and PPi. In the work, ispD gene from M. tuberculosis H37Rv (MtIspD) was cloned and expressed. With N-terminal fusion of a histidine-tagged sequence, MtIspD could be purified to homogeneity by one-step nickel affinity chromatography. MtIspD exists as a homodimer with an apparent molecular mass of 52 kDa. Enzyme property analysis revealed that MtIspD has high specificity for pyrimidine bases and narrow divalent cation requirements, with maximal activity found in the presence of CTP and Mg(2+). The turnover number of MtIspD is 3.4 s(-1). The Km for MEP and CTP are 43 and 92 muM, respectively. Furthermore, MtIspD shows thermal instable above 50 degrees C. Circular dichroism spectra revealed that the alteration of tertiary conformation is closely related with sharp loss of enzyme activity at higher temperature. This study is expected to help better understand the features of IspD and provide useful information for the development of novel antibiotics to treat M. tuberculosis.     

Molecular cloning, expression and characterization of the first three genes in the mevalonate-independent isoprenoid pathway in Streptomyces coelicolor.

The mevalonate-independent biosynthetic pathway to isopentenyl diphosphate and dimethylallyl diphosphate, the universal precursors to the isoprenoids, operates in eubacteria, including Escherichia coli, in algae, and in the plastids of higher plants. A search of the Sanger Centre Streptomyces coelicolor genome database revealed open reading frames with ca. 40--50% identity at the deduced amino acid level to the first three E. coli enzymes of this pathway, corresponding to deoxyxylulose phosphate synthase, deoxyxylulose phosphate reductoisomerase and 2-C-methyl erythritol 4-phosphate cytidylyltransferase. The S. coelicolor genes have been cloned and expressed in E. coli, and the recombinant proteins characterized physically and kinetically. The presence of the corresponding enzyme activities in extracts of S. coelicolor CH999 further supports the operation of the mevalonate-independent pathway in this organism.     

Isoprenoid biosynthesis via 1-deoxy-D-xylulose 5-phosphate/2-C-methyl-D-erythritol 4-phosphate (DOXP/MEP) pathway

Higher plants, several algae, bacteria, some strains of Streptomyces and possibly malaria parasite Plasmodium falciparum contain the novel, plastidic DOXP/MEP pathway for isoprenoid biosynthesis. This pathway, alternative with respect to the classical mevalonate pathway, starts with condensation of pyruvate and glyceraldehyde-3-phosphate which yields 1-deoxy-D-xylulose 5-phosphate (DOXP); the latter product can be converted to isopentenyl diphosphate (IPP) and eventually to isoprenoids or thiamine and pyridoxal. Subsequent reactions of this pathway involve transformation of DOXP to 2-C-methyl-D-erythritol 4-phosphate (MEP) which after condensation with CTP forms 4-diphosphocytidyl-2-amethyl-D-erythritol (CDP-ME). Then CDP-ME is phosphorylated to 4-diphosphocytidyl-2-amethyl-D-erythritol 2-phosphate (CDP-ME2P) and to 2-C-methyl-D-erythritol-2,4-cyclodiphosphate (ME-2,4cPP) which is the last known intermediate of the DOXP/MEP pathway. For- mation of IPP and dimethylallyl diphosphate (DMAPP) from ME-2,4cPP still requires clarification. This novel pathway appears to be involved in biosynthesis of carotenoids, phytol (side chain of chlorophylls), isoprene, mono-, di-, tetraterpenes and plastoquinone whereas the mevalonate pathway is responsible for formation of sterols, sesquiterpenes and triterpenes. Several isoprenoids were found to be of mixed origin suggesting that some exchange and/or cooperation exists between these two pathways of different biosynthetic origin. Contradictory results described below could indicate that these two pathways are operating under different physiological conditions of the cell and are dependent on the developmental state of plastids.     

GcpE is involved in the 2-C-methyl-D-erythritol 4-phosphate pathway of isoprenoid biosynthesis in Escherichia coli

In a variety of organisms, including plants and several eubacteria, isoprenoids are synthesized by the mevalonate-independent 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway. Although different enzymes of this pathway have been described, the terminal biosynthetic steps of the MEP pathway have not been fully elucidated. In this work, we demonstrate that the gcpE gene of Escherichia coli is involved in this pathway. E. coli cells were genetically engineered to utilize exogenously provided mevalonate for isoprenoid biosynthesis by the mevalonate pathway. These cells were then deleted for the essential gcpE gene and were viable only if the medium was supplemented with mevalonate or the cells were complemented with an episomal copy of gcpE.     

Crystal structure of 1-deoxy-D-xylulose 5-phosphate synthase, a crucial enzyme for isoprenoids biosynthesis

Isopentenyl pyrophosphate (IPP) is a common precursor for the synthesis of all isoprenoids, which have important functions in living organisms. IPP is produced by the mevalonate pathway in archaea, fungi, and animals. In contrast, IPP is synthesized by a mevalonate-independent pathway in most bacteria, algae, and plant plastids. 1-Deoxy-D-xylulose 5-phosphate synthase (DXS) catalyzes the first and the rate-limiting step of the mevalonate-independent pathway and is an attractive target for the development of novel antibiotics, antimalarials, and herbicides. We report here the first structural information on DXS, from Escherichia coli and Deinococcus radiodurans, in complex with the coenzyme thiamine pyrophosphate (TPP). The structure contains three domains (I, II, and III), each of which bears homology to the equivalent domains in transketolase and the E1 subunit of pyruvate dehydrogenase. However, DXS has a novel arrangement of these domains as compared with the other enzymes, such that the active site of DXS is located at the interface of domains I and II in the same monomer, whereas that of transketolase is located at the interface of the dimer. The coenzyme TPP is mostly buried in the complex, but the C-2 atom of its thiazolium ring is exposed to a pocket that is the substrate-binding site. The structures identify residues that may have important roles in catalysis, which have been confirmed by our mutagenesis studies.     

Hexameric assembly of the bifunctional methylerythritol 2,4-cyclodiphosphate synthase and protein-protein associations in the deoxy-xylulose-dependent pathway of isoprenoid precursor biosynthesis

The bifunctional methylerythritol 4-phosphate cytidylyltransferase methylerythritol 2,4-cyclodiphosphate synthase (IspDF) is unusual in that it catalyzes nonconsecutive reactions in the 1-deoxy-D-xylulose 5-phosphate (DOXP) pathway of isoprenoid precursor biosynthesis. The crystal structure of IspDF from the bacterial pathogen Campylobacter jejuni reveals an elongated hexamer with D3 symmetry compatible with the dimeric 2C-methyl-D-erythritol-4-phosphate cytidylyltransferase and trimeric 2C-methyl-D-erythritol-2,4-cyclodiphosphate synthase monofunctional enzymes. Complex formation of IspDF with 4-diphosphocytidyl-2C-methyl-D-erythritol kinase (IspE), the intervening enzyme activity in the pathway, has been observed in solution for the enzymes from C. jejuni and Agrobacterium tumefaciens. The monofunctional enzymes (2C-methyl-D-erythritol-4-phosphate cytidylyltransferase, IspE, and 2C-methyl-D-erythritol-2,4-cyclodiphosphate synthase) involved in the DOXP biosynthetic pathway of Escherichia coli also show physical associations. We propose that complex formation of the three enzymes at the core of the DOXP pathway can produce an assembly localizing 18 catalytic centers for the early stages of isoprenoid biosynthesis.     

Development of the activities of enzymes of the isoprenoid pathway during early stages of pea-seed germination

The activities of individual enzymes of the isoprenoid pathway from mevalonate kinase to squalene synthetase in homogenates of seeds germinated up to 32h were assayed. Changes in the activity of each enzyme were observed and compared with the activity at the 2h germination stage. Activities of alkaline phosphatase and fructose 1,6-diphosphate aldolase were similarly measured to provide a reference for changes in the general metabolic activity of seeds during imbibition of water. Water uptake reached a plateau after 12h. The reference enzymes almost doubled in activity between 2 and 8h and thereafter their activities steadily declined. All of the enzymes of the isoprenoid pathway increased in activity between 2 and 6h and, thereafter, with the exception of the prenyltransferase, their activities remained relatively constant. With the prenyltransferase activity the initial increase was followed by a short plateau between 6 and 9h and then a second increase to a maximum between 14 and 16h. After 16h the activity declined. The relative activities of the isoprenoid enzymes at 16h of germination were mevalonate kinase>phosphomevalonate kinase>pyrophosphomevalonate decarboxylase≈isopentenyl pyrophosphate isomerase>squalene synthetase>isopentenyl pyrophosphate/dimethylallyl pyrophosphate prenyltransferase. The finding that the prenyltransferase may be the rate-limiting enzyme in squalene synthesis from mevalonate is discussed in relation to regulation of isoprenoid synthesis during pea-seed germination.     

Structure of 2C-methyl-d-erythritol-2,4-cyclodiphosphate synthase involved in mevalonate-independent biosynthesis of isoprenoids

Isoprenoids are biosynthesized from isopentenyl diphosphate and the isomeric dimethylallyl diphosphate via the mevalonate pathway or a mevalonate-independent pathway that was identified during the last decade. The non-mevalonate pathway is present in many bacteria, some algae and in certain protozoa such as the malaria parasite Plasmodium falciparum and in the plastids of higher plants, but not in mammals and archaea. Therefore, these enzymes have been recognised as promising drug targets. We report the crystal structure of Escherichia coli 2C- methyl-d-erythritol-2,4-cyclodiphosphate synthase (IspF), which converts 4-diphosphocytidyl-2C-methyl-d-erythritol 2-phosphate into 2C-methyl-d-erythritol 2,4-cyclodiphosphate and CMP in a Mg-dependent reaction. The protein forms homotrimers that tightly bind one zinc ion per subunit at the active site, which helps to position the substrate for direct attack of the 2-phosphate group on the beta-phosphate.     

Production of mevalonate by a metabolically-engineered Escherichia coli

Mevalonate is biosynthesized from acetyl-CoA and metabolized to isoprenoid compounds in a wide variety of organisms although certain types of prokaryotes employ another route for isoprenoid biosynthesis (the non-mevalonate pathway). To establish a fermentative process for mevalonate production, enzymes for mevalonate synthesis from Enterococcus faecalis were expressed in Escherichia coli, a non-mevalonate pathway bacterium. Mevalonate was accumulated, indicating a redirection of acetate metabolism by the expressed enzyme. The recombinant E. coli produced 47 g mevalonate l(-1) in 50 h of fed-batch cultivation in a 2 l jar fermenter; this is the highest titer ever reported demonstrating the superiority of E. coli in its ability of acetyl-CoA supply and its inability is degrade mevalonate.     

Cloning and expression of IspDF from Mesorhizobium loti. Characterization of a bifunctional protein that catalyzes non-consecutive steps in the methylerythritol phosphate pathway

Gram-negative bacteria, plant chloroplasts, green algae and some Gram-positive bacteria utilize the 2-C-methyl-d-erythritol phosphate (MEP) pathway for the biosynthesis of isoprenoids. IspD, ispE, and ispF encode the enzymes required to convert MEP to 2-C-methyl-d-erythritol 2,4-cyclodiphosphate (cMEDP) during the biosynthesis of isopentenyl diphosphate and dimethylallyl diphosphate in the MEP pathway. Upon analysis of the Mesorhizobium loti genome, ORF mll0395 showed homology to both ispD and ispF and appeared to encode a fusion protein. M. loti ispE was located elsewhere on the chromosome. Purified recombinant IspDF protein was mostly a homodimer, MW approximately 46 kDa/subunit. Incubation of IspDF with MEP, CTP, and ATP gave 4-diphosphocytidyl-2-C-methyl-d-erythritol (CDP-ME) as the only product. When Escherichia coli IspE protein was added to the incubation mixture, cMEDP was formed. In addition, M. loti ORF mll0395 complements lethal disruptions in both ispD and ispF in Salmonella typhimurium. These results indicate that IspDF is a bifunctional protein, which catalyzes the first and third steps in the conversion of MEP to cMEDP.     

Structural similarities in the noncatalytic domains of phenylalanyl-tRNA and biotin synthetases.

Detailed comparison between the structures of the Escherichia coli biotin synthetase/repressor protein (BirA) and the recently solved Thermus thermophilus phenylalanyl-tRNA synthetase (PheRS) reveals significant similarities outside their respective catalytic domains. These comprise a DNA-binding alpha+beta domain and an Src-homology 3 (SH3)-like domain that were observed in both enzymes. This similarity provides a novel example in which all domains of one multidomain protein appear to be constituents of the other multidomain protein and supports a concept of a common ancestor for two different synthetase families.