(1/54) Biosynthesis of terpenoids: YchB protein of Escherichia coli phosphorylates the 2-hydroxy group of 4-diphosphocytidyl-2C-methyl-D-erythritol.
A comparative analysis of all published complete genomes indicated that the putative orthologs of the unannotated ychB gene of Escherichia coli follow the distribution of the dxs, dxr, and ygbP genes, which have been shown to specify enzymes of the deoxyxylulose phosphate pathway of terpenoid biosynthesis, thus suggesting that the hypothetical YchB protein also is involved in that pathway. To test this hypothesis, the E. coli ychB gene was expressed in a homologous host. The recombinant protein was purified to homogeneity and was shown to phosphorylate 4-diphosphocytidyl-2C-methyl-D-erythritol in an ATP-dependent reaction. The reaction product was identified as 4-diphosphocytidyl-2C-methyl-D-erythritol 2-phosphate by NMR experiments with various (13)C-labeled substrate samples. A (14)C-labeled specimen of this compound was converted efficiently into carotenoids by isolated chromoplasts of Capsicum annuum. The sequence of E. coli YchB protein is similar to that of the protein predicted by the tomato cDNA pTOM41 (30% identity), which had been implicated in the conversion of chloroplasts to chromoplasts. (+info)
(2/54) Regulation of expression of the yiaKLMNOPQRS operon for carbohydrate utilization in Escherichia coli: involvement of the main transcriptional factors.
The yiaKLMNOPQRS (yiaK-S) gene cluster of Escherichia coli is believed to be involved in the utilization of a hitherto unknown carbohydrate which generates the intermediate L-xylulose. Transcription of yiaK-S as a single message from the unique promoter found upstream of yiaK is proven in this study. The 5' end has been located at 60 bp upstream from the ATG. Expression of the yiaK-S operon is controlled in the wild-type strain by a repressor encoded by yiaJ. No inducer molecule of the yiaK-S operon has been identified among over 80 carbohydrate or derivative compounds tested, the system being expressed only in a mutant strain lacking the YiaJ repressor. The lacZ transcriptional fusions in the genetic background of the mutant strain revealed that yiaK-S is modulated by the integration host factor and by the cyclic AMP (cAMP)-cAMP receptor protein (Crp) activator complex. A twofold increase in the induction was observed during anaerobic growth, which was independent of ArcA or Fnr. Gel mobility shift assays showed that the YiaJ repressor binds to a promoter fragment extending from -50 to +121. These studies also showed that the cAMP-Crp complex can bind to two different sites. The lacZ transcriptional fusions of different fragments of the promoter demonstrated that binding of cAMP-Crp to the Crp site 1, centered at -106, is essential for yiaK-S expression. The 5' end of the yiaJ gene was determined, and its promoter region was found to overlap with the divergent yiaK-S promoter. Expression of yiaJ is autogenously regulated and reduced by the binding of Crp-cAMP to the Crp site 1 of the yiaK-S promoter. (+info)
(3/54) Role of the yiaR and yiaS genes of Escherichia coli in metabolism of endogenously formed L-xylulose.
Genes yiaP and yiaR of the yiaKLMNOPQRS cluster of Escherichia coli are required for the metabolism of the endogenously formed L-xylulose, whereas yiaS is required for this metabolism only in araD mutants. Like AraD, YiaS was shown to have L-ribulose-5-phosphate 4-epimerase activity. Similarity of YiaR to several 3-epimerases suggested that this protein could catalyze the conversion of L-xylulose-5-phosphate into L-ribulose-5-phosphate, thus completing the pathway between L-xylulose and the general metabolism. (+info)
(4/54) Phosphorylation of 1-deoxy-D-xylulose by D-xylulokinase of Escherichia coli.
1-deoxy-D-xylulose 5-phosphate serves as a precursor for the biosynthesis of the vitamins thiamine and pyridoxal and for the formation of isopentenyl pyrophosphate and dimethylallyl pyrophosphate via the nonmevalonate pathway of terpenoid biosynthesis. Earlier studies had shown that Escherichia coli incorporates unphosphorylated 1-deoxy-D-xylulose into the terpenoid side chain of ubiquinones with high efficacy. We show that D-xylulokinase of E. coli (EC 220.127.116.11) catalyzes the phosphorylation of 1-deoxy-D-xylulose at the hydroxy group of C-5 at a rate of 1.6 micromol.mg min-1. This reaction constitutes a potential salvage pathway for the generation of 1-deoxy-D-xylulose 5-phosphate from exogenous or endogenous 1-deoxy-D-xylulose as starting material for the biosynthesis of terpenoids, thiamine and pyridoxal. (+info)
(5/54) The plastidic pentose phosphate translocator represents a link between the cytosolic and the plastidic pentose phosphate pathways in plants.
Plastids are the site of the reductive and the oxidative pentose phosphate pathways, which both generate pentose phosphates as intermediates. A plastidic transporter from Arabidopsis has been identified that is able to transport, in exchange with inorganic phosphate or triose phosphates, xylulose 5-phosphate (Xul-5-P) and, to a lesser extent, also ribulose 5-phosphate, but does not accept ribose 5-phosphate or hexose phosphates as substrates. Under physiological conditions, Xul-5-P would be the preferred substrate. Therefore, the translocator was named Xul-5-P/phosphate translocator (XPT). The XPT shares only approximately 35% to 40% sequence identity with members of both the triose phosphate translocator and the phosphoenolpyruvate/phosphate translocator classes, but a higher identity of approximately 50% to glucose 6-phosphate/phosphate translocators. Therefore, it represents a fourth group of plastidic phosphate translocators. Database analysis revealed that plant cells contain, in addition to enzymes of the oxidative branch of the oxidative pentose phosphate pathway, ribose 5-phosphate isomerase and ribulose 5-phosphate epimerase in both the cytosol and the plastids, whereas the transketolase and transaldolase converting the produced pentose phosphates to triose phosphates and hexose phosphates are probably solely confined to plastids. It is assumed that the XPT function is to provide the plastidic pentose phosphate pathways with cytosolic carbon skeletons in the form of Xul-5-P, especially under conditions of a high demand for intermediates of the cycles. (+info)
(6/54) Molecular cloning of XYL3 (D-xylulokinase) from Pichia stipitis and characterization of its physiological function.
XYL3, which encodes a D-xylulokinase (EC 18.104.22.168), was isolated from Pichia stipitis CBS 6054 genomic DNA by using primers designed against conserved motifs. Disruption of XYL3 eliminated D-xylulokinase activity, but D-ribulokinase activity was still present. Southern analysis of P. stipitis genomic DNA with XYL3 as a probe confirmed the disruption and did not reveal additional related genes. Disruption of XYL3 stopped ethanol production from xylose, but the resulting mutant still assimilated xylose slowly and formed xylitol and arabinitol. These results indicate that XYL3 is critical for ethanol production from xylose but that P. stipitis has another pathway for xylose assimilation. Expression of XYL3 using its P. stipitis promoter increased Saccharomyces cerevisiae D-xylulose consumption threefold and enabled the transformants to produce ethanol from a mixture of xylose and xylulose, whereas the parental strain only accumulated xylitol. In vitro, D-xylulokinase activity in recombinant S. cerevisiae was sixfold higher with a multicopy than with a single-copy XYL3 plasmid, but ethanol production decreased with increased copy number. These results confirmed the function of XYL3 in S. cerevisiae. (+info)
(7/54) Biosynthesis of vitamin B6 in Rhizobium: in vitro synthesis of pyridoxine from 1-deoxy-D-xylulose and 4-hydroxy-L-threonine.
Pyridoxine (vitamin B6) in Rhizobium is synthesized from 1-deoxy-D-xylulose and 4-hydroxy-L-threonine. To define the pathway enzymatically, we established an enzyme reaction system with a crude enzyme solution of R. meliloti IFO14782. The enzyme reaction system required NAD+, NADP+, and ATP as coenzymes, and differed from the E. coli enzyme reaction system comprising PdxA and PdxJ proteins, which requires only NAD+ for formation of pyridoxine 5'-phosphate from 1-deoxy-D-xylulose 5-phosphate and 4-(phosphohydroxy)-L-threonine. (+info)
(8/54) Contribution of the mevalonate and methylerythritol phosphate pathways to the biosynthesis of gibberellins in Arabidopsis.
Gibberellins (GAs) are diterpene plant hormones essential for many developmental processes. Although the GA biosynthesis pathway has been well studied, our knowledge on its early stage is still limited. There are two possible routes for the biosynthesis of isoprenoids leading to GAs, the mevalonate (MVA) pathway in the cytosol and the methylerythritol phosphate (MEP) pathway in plastids. To distinguish these possibilities, metabolites from each isoprenoid pathway were selectively labeled with (13)C in Arabidopsis seedlings. Efficient (13)C-labeling was achieved by blocking the endogenous pathway chemically or genetically during the feed of a (13)C-labeled precursor specific to the MVA or MEP pathways. Gas chromatography-mass spectrometry analyses demonstrated that both MVA and MEP pathways can contribute to the biosyntheses of GAs and campesterol, a cytosolic sterol, in Arabidopsis seedlings. While GAs are predominantly synthesized through the MEP pathway, the MVA pathway plays a major role in the biosynthesis of campesterol. Consistent with some crossover between the two pathways, phenotypic defects caused by the block of the MVA and MEP pathways were partially rescued by exogenous application of the MEP and MVA precursors, respectively. We also provide evidence to suggest that the MVA pathway still contributes to GA biosynthesis when this pathway is limiting. (+info)
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