The localisation of 2-carboxy-D-arabinitol 1-phosphate and inhibition of Rubisco in leaves of Phaseolus vulgaris L. (1/353)

A recent controversial report suggests that the nocturnal inhibitor of Rubisco, 2-carboxy-D-arabinitol 1-phosphate (CAIP), does not bind to Rubisco in vivo and therefore that CA1P has no physiological relevance to photosynthetic regulation. It is now proved that a direct rapid assay can be used to distinguish between Rubisco-bound and free CA1P, as postulated in the controversial report. Application of this direct assay demonstrates that CA1P is bound to Rubisco in vivo in dark-adapted leaves. Furthermore, CA1P is shown to be in the chloroplasts of mesophyll cells. Thus, CA1P does play a physiological role in the regulation of Rubisco.  (+info)

Expression of prokaryotic 1-deoxy-D-xylulose-5-phosphatases in Escherichia coli increases carotenoid and ubiquinone biosynthesis. (2/353)

Isopentenyl diphosphate (IPP) acts as the common, five-carbon building block in the biosynthesis of all isoprenoids. The first reaction of IPP biosynthesis in Escherichia coli is the formation of 1-deoxy-D-xylulose-5-phosphate, catalysed by 1-deoxy-D-xylulose-5-phosphate synthase (DXPS). E. coli engineered to produce lycopene, was transformed with dxps genes cloned from Bacillus subtilis and Synechocystis sp. 6803. Increases in lycopene levels were observed in strains expressing exogenous DXPS compared to controls. The recombinant strains also exhibited elevated levels of ubiquinone-8. These increases corresponded with enhanced DXP synthase activity in the recombinant E. coli strains.  (+info)

Vitamin B6 biosynthesis: formation of pyridoxine 5'-phosphate from 4-(phosphohydroxy)-L-threonine and 1-deoxy-D-xylulose-5-phosphate by PdxA and PdxJ protein. (3/353)

In Escherichia coli the coenzyme pyridoxal 5'-phosphate (PLP) is synthesised de novo by a pathway that is thought to involve the condensation of 4-(phosphohydroxy)-L-threonine and 1-deoxy-D-xylulose, catalysed by the enzymes PdxA and PdxJ, to form either pyridoxine (vitamin B6) or pyridoxine 5'-phosphate (PNP). Here we show that incubation of PdxJ with PdxA, 4-(phosphohydroxy)-L-threonine, NAD and 1-deoxy-D-xylulose-5-phosphate, but not 1-deoxy-D-xylulose, results in the formation of PNP. The PNP formed was characterised by (i) cochromatography with an authentic standard, (ii) conversion to pyridoxine by alkaline phosphatase treatment, and (iii) UV and fluorescence spectroscopy. Furthermore, when [2-(14)C]1-deoxy-D-xylulose-5-phosphate was used as a substrate, the radioactivity was incorporated into PNP. These results clarify the previously unknown role of PdxJ in the de novo PLP biosynthetic pathway. The sugar used as substrate by PdxJ is 1-deoxy-D-xylulose-5-phosphate rather than the previously assumed 1-deoxy-D-xylulose. The first vitamin B6 vitamer synthesised is PNP, and not pyridoxine.  (+info)

Crystal structure of carboxylase reaction-oriented ribulose 1, 5-bisphosphate carboxylase/oxygenase from a thermophilic red alga, Galdieria partita. (4/353)

Ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco, EC 4.1.1. 39) obtained from a thermophilic red alga Galdieria partita has the highest specificity factor of 238 among the Rubiscos hitherto reported. Crystal structure of activated Rubisco from G. partita complexed with the reaction intermediate analogue, 2-carboxyarabinitol 1,5-bisphosphate (2-CABP) has been determined at 2.4-A resolution. Compared with other Rubiscos, different amino residues bring the structural differences in active site, which are marked around the binding sites of P-2 phosphate of 2-CABP. Especially, side chains of His-327 and Arg-295 show the significant differences from those of spinach Rubisco. Moreover, the side chains of Asn-123 and His-294 which are reported to bind the substrate, ribulose 1,5-bisphosphate, form hydrogen bonds characteristic of Galdieria Rubisco. Small subunits of Galdieria Rubisco have more than 30 extra amino acid residues on the C terminus, which make up a hairpin-loop structure to form many interactions with the neighboring small subunits. When the structures of Galdieria and spinach Rubiscos are superimposed, the hairpin region of the neighboring small subunit in Galdieria enzyme and apical portion of insertion residues 52-63 characteristic of small subunits in higher plant enzymes are almost overlapped to each other.  (+info)

Construction and characterization of Escherichia coli disruptants defective in the yaeM gene. (5/353)

Escherichia coli disruptants defective in the yaeM gene, which is located at 4.2 min on the chromosome map, were constructed and characterized. The disruptants showed auxotrophy for 2-C-methylerythritol, a free alcohol of 2-C-methyl-D-erythritol 4-phosphate that is a biosynthetic precursor in the nonmevalonate pathway. This result clearly shows that the yaeM gene is indeed involved in this pathway in E. coli.  (+info)

Inhibitors of the nonmevalonate pathway of isoprenoid biosynthesis as antimalarial drugs. (6/353)

A mevalonate-independent pathway of isoprenoid biosynthesis present in Plasmodium falciparum was shown to represent an effective target for chemotherapy of malaria. This pathway includes 1-deoxy-D-xylulose 5-phosphate (DOXP) as a key metabolite. The presence of two genes encoding the enzymes DOXP synthase and DOXP reductoisomerase suggests that isoprenoid biosynthesis in P. falciparum depends on the DOXP pathway. This pathway is probably located in the apicoplast. The recombinant P. falciparum DOXP reductoisomerase was inhibited by fosmidomycin and its derivative, FR-900098. Both drugs suppressed the in vitro growth of multidrug-resistant P. falciparum strains. After therapy with these drugs, mice infected with the rodent malaria parasite P. vinckei were cured.  (+info)

A Synechococcus leopoliensis SAUG 1402-1 operon harboring the 1-deoxyxylulose 5-phosphate synthase gene and two additional open reading frames is functionally involved in the dimethylallyl diphosphate synthesis. (7/353)

Experiments have been performed to prove the existence and the functionality of the novel mevalonate independent 1-deoxyxylulose 5-phosphate isoprenoid biosynthesis pathway in cyanobacteria. For this purpose, a segment of the 1-deoxyxylulose 5-phosphate synthase gene (dxs) was amplified from Synechococcus leopoliensis SAUG 1402-1 DNA via PCR using oligonucleotides for conserved regions of dxs. Subsequent hybridization screening of a genomic cosmid library of S. leopoliensis with this segment has led to the identification of an 18.7 kbp segment of the S. leopoliensis genome on which a dxs homologous gene and two adjacent open reading frames organized in one operon could be localized by DNA sequencing. The three genes of the operon were separately expressed in Escherichia coli, proving that the identified cyanobacterial dxs is functionally involved in the formation of dimethylallyl diphosphate, one basic intermediate of isoprenoid biosynthesis.  (+info)

2'-carboxy-D-arabitinol 1-phosphate protects ribulose 1, 5-bisphosphate carboxylase/oxygenase against proteolytic breakdown. (8/353)

Trypsin-catalysed cleavage of purified ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) and the resultant irreversible loss of carboxylase activity were prevented by prior incubation with the naturally occurring nocturnal Rubisco inhibitor 2'-carboxy-D-arabitinol 1-phosphate (CA1P), as well as with ribulose 1,5-bisphosphate (RuBP), Mg2+ and CO2. CA1P also protected Rubisco from loss of activity caused by carboxypeptidase A. When similar experiments were carried out using soluble chloroplast proteases, CA1P was again able to protect Rubisco against proteolytic degradation and the consequent irreversible loss of catalytic activity. Thus, CA1P prevents the proteolytic breakdown of Rubisco by endogenous and exogenous proteases. In this way, CA1P may affect the amounts of Rubisco protein available for photosynthetic CO2 assimilation. Rubisco turnover (in the presence of RuBP, Mg2+ and CO2) may confer similar protection against proteases in the light.  (+info)