A fluorometric assay for the determination of 1-deoxy-D-xylulose 5-phosphate synthase activity

We report a novel fluorometric end-point assay for the determination of 1-deoxy-d-xylulose 5-phosphate synthase (DXS) activity based on the reaction of 1-deoxy-D-xylulose 5-phosphate (DX5P) with 3,5-diaminobenzoic acid in an acidic medium to form a highly fluorescent quinaldine derivative. The assay was validated in three ways: (a) for a fixed amount of DXS in the reaction mixture the emitted fluorescence increased linearly with the reaction time, (b) for a fixed reaction time fluorescence intensity increased with the concentration of DXS in the reaction mixture, and (c) the increase in fluorescence intensity correlated (r = 0.99; P < 0.002) with the amount of DX5P formed in the reaction mixture determined radiometrically. The sensitivity of the fluorometric assay is similar to that of the previously described radiometric methods. This assay can be useful for the functional characterization of DXS as well as for the screening of DXS inhibitors with potential antibiotic, herbicidal, or antimalarial action.     

冬凌草1-脱氧-D-木酮糖-5-磷酸合成酶基因克隆与表达分析

1-脱氧-D-木酮糖-5-磷酸合成酶(1-deoxy-D-xylulose 5-phosphate synthase,DXS)是植物萜类代谢通路中2-C-甲基-D-赤藓糖醇-4-磷酸(MEP)途径的第一个关键酶,在植物萜类物质的生物合成中发挥重要的作用.为了研究该基因在冬凌草二萜类成分合成中的作用,该研究在冬凌草转录组测序结果的基础上设计一对特异性引物,采用RT-PCR方法得到冬凌草IrDXS基因cDNA全长序列,并对其蛋白进行理化性质分析、信号肽预测、亚细胞定位预测、蛋白质二级结构、三级结构预测分析及跨膜域分析等生物信息学分析,同时利用实时荧光定量PCR的方法检测IrDXS基因在冬凌草不同部位中的表达情况.结果表明:从冬凌草叶片中分离得到了一条编码DXS的全长基因,通过生物信息学软件分析发现,该基因编码全长2169 bp,编码722个氨基酸,分子量为77.7 kD.多序列比对发现该基因编码的蛋白和其他植物中已知的DXS蛋白序列具有较高的同源性,N端均包含了一段质体转运肽序列,并均具有一个保守的焦磷酸硫胺素结构域和与吡啶结合相关的DRAG结构域.序列进化树分析显示,IrDXS基因属于植物DXS2家族.DXS基因在冬凌草根中表达量最高、愈伤组织中最低.该研究首次获得了IrDXS基因的全长cDNA序列,并揭示了其在不同组织中的表达差异,为后续的深入研究IrDXS基因在冬凌草二萜类成分合成途径中的功能奠定了基础.     

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.     

A role for symplastic gating in the control of the potato tuber life cycle

The control of the potato tuber life cycle has been the subject of significant interest over many years. A number of different approaches have been adopted and data is available regarding hormonal, metabolic and gene expression changes that occur over the tuber life cycle. Despite this intense effort, no unifying model for the control of the potato tuber life cycle has emerged. We have undertaken a detailed analysis of the tuber life cycle utilising physiological, biochemical and cell-biological techniques. It has emerged that a major factor contributing to both tuber induction and dormancy break is symplastic gating which controls the allocation of resources to meristematic or vegetative tissues. Future challenges include the determination of factors regulating symplastic gating at the molecular level and the extrapolation of these findings to other systems.Key words: development, tuber, potato     

1-Deoxy-D-xylulose-5-phosphate synthase, a limiting enzyme for plastidic isoprenoid biosynthesis in plants

The initial step of the plastidic 2C-methyl-D-erythritol 4-phosphate (MEP) pathway that produces isopentenyl diphosphate is catalyzed by 1-deoxy-d-xylulose-5-phosphate synthase. To investigate whether or not 1-deoxy-d-xylulose-5-phosphate synthase catalyzes a limiting step in the MEP pathway in plants, we produced transgenic Arabidopsis plants that over- or underexpress this enzyme. Compared with non-transgenic wild-type plants, the transgenic plants accumulate different levels of various isoprenoids such as chlorophylls, tocopherols, carotenoids, abscisic acid, and gibberellins. Phenotypically, the transgenic plants had slight alterations in growth and germination rates. Because the levels of several plastidic isoprenoids correlate with changes in 1-deoxy-D-xylulose-5-phosphate synthase levels, we conclude that this enzyme catalyzes one of the rate-limiting steps of the MEP biosynthetic pathway. Furthermore, since the product of the MEP pathway is isopentenyl diphosphate, our results suggest that in plastids the pool of isopentenyl diphosphate is limiting to isprenoid production.     

Carotenoid biosynthesis during tomato fruit development: regulatory role of 1-deoxy-D-xylulose 5-phosphate synthase

Plant isoprenoids represent a heterogeneous group of compounds which play essential roles not only in growth and development, but also in the interaction of plants with their environment. Higher plants contain two pathways for the biosynthesis of isoprenoids: the mevalonate pathway, located in the cytosol/endoplasmic reticulum, and the recently discovered mevalonate-independent pathway (Rohmer pathway), located in the plastids. In order to evaluate the function of the Rohmer pathway in the regulation of the synthesis of plastidial isoprenoids, we have isolated a tomato cDNA encoding 1-deoxy-D-xylulose 5-phosphate synthase (DXS), the first enzyme of the pathway. We demonstrate in vivo activity and plastid targeting of plant DXS. Expression analysis of the tomato DXS gene indicates developmental and organ-specific regulation of mRNA accumulation and a strong correlation with carotenoid synthesis during fruit development. 1-Deoxy-D-xylulose feeding experiments, together with expression analysis of DXS and PSY1 (encoding the fruit-specific isoform of phytoene synthase) in wild-type and yellow flesh mutant fruits, indicate that DXS catalyses the first potentially regulatory step in carotenoid biosynthesis during early fruit ripening. Our results change the current view that PSY1 is the only regulatory enzyme in tomato fruit carotenogenesis, and point towards a coordinated role of both DXS and PSY1 in the control of fruit carotenoid synthesis.     

Essential role of residue H49 for activity of Escherichia coli 1-deoxy-D-xylulose 5-phosphate synthase, the enzyme catalyzing the first step of the 2-C-methyl-D-erythritol 4-phosphate pathway for isoprenoid Synthesis.

The first step of the 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway for isoprenoid biosynthesis in plant plastids and most eubacteria is catalyzed by 1-deoxy-D-xylulose 5-phosphate synthase (DXS), a recently described transketolase-like enzyme. To identify key residues for DXS activity, we compared the amino acid sequence of Escherichia coli DXS with that of E. coli and yeast transketolase (TK). Alignment showed a previously undetected conserved region containing an invariant histidine residue that has been described to participate in proton transfer during TK catalysis. The possible role of the conserved residue in E. coli DXS (H49) was examined by site-directed mutagenesis. Replacement of this histidine residue with glutamine yielded a mutant DXS-H49Q enzyme that showed no detectable DXS activity. These findings are consistent with those obtained for yeast TK and demonstrate a key role of H49 for DXS activity.     

Functional and evolutionary analysis of DXL1, a non-essential gene encoding a 1-deoxy-D-xylulose 5-phosphate synthase like protein in Arabidopsis thaliana

The synthesis of 1-deoxy-D-xylulose 5-phosphate (DXP), catalyzed by the enzyme DXP synthase (DXS), represents a key regulatory step of the 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway for isoprenoid biosynthesis. In plants DXS is encoded by small multigene families that can be classified into, at least, three specialized subfamilies. Arabidopsis thaliana contains three genes encoding proteins with similarity to DXS, including the well-known DXS1/CLA1 gene, which clusters within subfamily I. The remaining proteins, initially named DXS2 and DXS3, have not yet been characterized. Here we report the expression and functional analysis of A. thaliana DXS2. Unexpectedly, the expression of DXS2 failed to rescue Escherichia coli and A. thaliana mutants defective in DXS activity. Coherently, we found that DXS activity was negligible in vitro, being renamed as DXL1 following recent nomenclature recommendation. DXL1 is targeted to plastids as DXS1, but shows a distinct expression pattern. The phenotypic analysis of a DXL1 defective mutant revealed that the function of the encoded protein is not essential for growth and development. Evolutionary analyses indicated that DXL1 emerged from DXS1 through a recent duplication apparently specific of the Brassicaceae lineage. Divergent selective constraints would have affected a significant fraction of sites after diversification of the paralogues. Furthermore, amino acids subjected to divergent selection and likely critical for functional divergence through the acquisition of a novel, although not yet known, biochemical function, were identified. Our results provide with the first evidences of functional specialization at both the regulatory and biochemical level within the plant DXS family.     

Isolation of the dxr gene of Zymomonas mobilis and characterization of the 1-deoxy-D-xylulose 5-phosphate reductoisomerase

The gene encoding the second enzyme of the 2C-methyl-D-erythritol 4-phosphate (MEP) pathway for isopentenyl diphosphate biosynthesis, 1-deoxy-D-xylulose 5-phosphate (DXP) reductoisomerase, was cloned and sequenced from Zymomonas mobilis. The deduced amino acid sequence showed the highest identity (48.2%) to the DXP reductoisomerase of Escherichia coli. Biochemical characterization of the purified DXP reductoisomerase showed a strict dependence of the enzyme on NADPH and divalent cations (Mn(2+), Co(2+) or Mg(2+)). The enzyme is a dimer with a molecular mass of 39 kDa per subunit and has a specific activity of 19.5 U mg protein(-1). Catalysis of the intramolecular rearrangement and reduction of DXP to MEP is competitively inhibited by the antibiotic fosmidomycin with a K(i) of 0.6 microM.     

A mutant pyruvate dehydrogenase E1 subunit allows survival of Escherichia coli strains defective in 1-deoxy-D-xylulose 5-phosphate synthase

The 2-C-methyl-D-erythritol 4-phosphate pathway has been proposed as a promising target to develop new antimicrobial agents. However, spontaneous mutations in Escherichia coli were observed to rescue the otherwise lethal loss of the first two enzymes of the pathway, 1-deoxy-D-xylulose 5-phosphate (DXP) synthase (DXS) and DXP reductoisomerase (DXR), with a relatively high frequency. A mutation in the gene encoding the E1 subunit of the pyruvate dehydrogenase complex was shown to be sufficient to rescue the lack of DXS but not DXR in vivo, suggesting that the mutant enzyme likely allows the synthesis of DXP or an alternative substrate for DXR.     

Crystal structure of 1-deoxy-D-xylulose 5-phosphate reductoisomerase complexed with cofactors: implications of a flexible loop movement upon substrate binding

The key enzyme in the nonmevalonate pathway, 1-deoxy-D-xylulose 5-phosphate reductoisomerase (DXR), has been shown to be an effective target of antimalarial drugs. Here we report the crystal structure of DXR complexed with NADPH and a sulfate ion from Escherichia coli at 2.2 A resolution. The structure showed the presence of an extra domain, which is absent from other NADPH-dependent oxidoreductases, in addition to the conformation of catalytic residues and the substrate binding site. A flexible loop covering the substrate binding site plays an important role in the enzymatic reaction and the determination of substrate specificity.     

Evaluation of potato tuber moth (Lepidoptera: Gelechiidae) resistance in tubers of Bt-cry5 transgenic potato lines

The potato tuber moth, Phthorimaea operculella (Zeller), in tropical and subtropical countries, is the most destructive pest of potato, Solanum tuberosum L. The larvae attack foliage and tubers in the field and in storage. The purpose of this study was to evaluate the efficacy of a Bt-cry5 transgene to control the potato tuber moth in tuber tissues. Tuber bioassays using stored (11-12 mo old) and newly harvested tubers of Bt-cry5-Lemhi Russet and Bt-cry5-Atlantic potato lines showed up to 100% mortality of 1st instars. Mortality was lowest in the newly harvested tubers of Bt-cry5-Atlantic lines (47.1-67.6%). Potato tuber moth mortality was 100% in the Bt-cry5-Spunta lines that were transformed with Bt-cry5 gene controlled by the CaMV 35S promoter (pBIML5 vector) and in 2 of 3 lines transformed with Bt-cry5 gene controlled by the Gelvin super promoter (pBIML1 vector). The transgenic Spunta lines expressing Bt-cry5 controlled by the patatin promoter (pBMIL2 vector) showed the lowest tuber moth mortality (25.6 and 31.1%). The Bt-cry5 transgenic lines with high tuber expression of B. thuringiensis have value in an integrated pest management system to control potato tuber moth.     

A high-throughput screening assay for simultaneous selection of inhibitors of Mycobacterium tuberculosis 1-deoxy-D-xylulose-5-phosphate synthase (Dxs) or 1-deoxy-D-xylulose 5-phosphate reductoisomerase (Dxr)

1-deoxy-D-xylulose 5-phosphate reductoisomerase (Dxr) is involved in the synthesis of isoprenoids by the methylerythritol phosphate pathway. Dxr is essential in Mycobacterium tuberculosis (Mtu), absent in humans and amenable to structure-aided design. To further assess the druggability of the enzyme, the energetics of binding of fosmidomycin to Mtu Dxr was studied by isothermal calorimetry. Binding was enhanced by nicotinamide adenine dinucleotide phosphate hydrogen (NADPH) and driven by enthalpy (ΔH -10.2 kcal/mol, ΔS 1.1 cal mol(-1)K(-1)). This suggests the possibility of finding novel inhibitors that bind enthalpically, making Dxr an attractive target. The cost of the Dxr substrate, 1-deoxy-D-xylulose-5-phosphate, for high-throughput screening (HTS) is prohibitive. Hence, an HTS assay that couples Dxr to the upstream enzyme 1-deoxy-D-xylulose-5-phosphate synthase (Dxs), also a valid target, was developed. A high concentration of NADPH was used to bias it to detect Dxr inhibitors that bind like fosmidomycin. The assay Z' was 0.75. It was equally sensitive to inhibitors of Dxs and Dxr, that is, fosmidomycin and fluropyruvate inhibited it with IC(50)s similar to that in the individual enzyme assays (79 vs 54 nM for fosmidomycin). To distinguish inhibitors of Dxs from Dxr, individual enzyme assays and a microplate thermofluor binding assay were developed. The assay simultaneously screens two targets and is cost-effective.     

Temperature-sensitive Arabidopsis mutant defective in 1-deoxy-d-xylulose 5-phosphate synthase within the plastid non-mevalonate pathway of isoprenoid biosynthesis

The temperature-sensitive mutant of Arabidopsis , chs5 , developed chlorotic leaves at restrictive temperatures (15°C), but almost normal green leaves at permissive temperatures (22°C). At the restrictive temperature, the chs5 mutation blocked the accumulation of chlorophylls and carotenoids. A temperature-shift analysis revealed that the manifestation of the chlorotic phenotype occurred in young leaf tissues, but did not in mature leaf tissues. Genetic and sequence analysis demonstrated that the chs5 mutation was caused by a single-base change in the coding region of a recently identified CLA1 gene. The CLA1 gene exhibited a high sequence similarity to the genes encoding 1-deoxy- d -xylulose 5-phosphate synthase (DXS) localized to the non-mevalonate pathway, which was recently discovered in bacteria and higher plants. In addition, the application of 1-deoxy- d -xylulose, the free sugar of 1-deoxy- d -xylulose 5-phosphate, rescues the defect in the chs5 mutant. These results indicated that the chlorotic phenotype of the chs5 mutant was caused by a defect in DXS activity and that DXS functions preferentially at an early stage of leaf cell development. A transiently expressed green fluorescent protein fused with the CLA1 transit peptide was localized within the chloroplasts in the green cultured cells of tobacco, which suggests that the putative localization of the non-mevalonate pathway is in plastids.     

Mutation in the flexible loop of 1-deoxy-D-xylulose 5-phosphate reductoisomerase broadens substrate utilization

The second enzyme in the methylerythritol phosphate pathway to isoprenoids, 1-deoxy-D-xylulose 5-phosphate reductoisomerase (DXR; EC 1.1.1.267) mediates the transformation of 1-deoxy-D-xylulose 5-phosphate (DXP) into 2-C-methyl-D-erythritol 4-phosphate. Several DXR mutants have been prepared to study amino acid residues important in binding or catalysis, but in-depth studies of many conserved residues in the flexible loop portion of the enzyme have not been conducted. In the course of our studies of this enzyme, an analog of DXP, 1,2-dideoxy-D-threo-3-hexulose 6-phosphate (1-methyl-DXP), was found to be a weak competitive inhibitor. Using the X-ray crystal structures of DXR as a guide, a highly conserved tryptophan residue in the flexible loop was identified that potentially blocks the use of this analog as a substrate. To test this hypothesis, four mutants of the Synechocystis sp. PCC6803 DXR were prepared and a W204F mutant was found to utilize the analog as a substrate.