Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P06889 (Mol)
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We have studied the light-dependent expression of the Chlamydomonas reinhardtii csbp gene encoding sedoheptulose-1,7-bisphosphatase (SBPase), an enzyme of the pentose-phosphate pathway. Expression studies using light/dark-synchronized cultures revealed that csbp mRNA abundance increases significantly during illumination. We have used a 1.4 kb region upstream of the csbp gene in transcriptional fusions to the homologous arylsulfatase-encoding reporter gene (ars). In transformants carrying the chimeric csbp/ars reporter gene, arylsulfatase activity is detected in the absence of sulfate, a condition under which the endogenous ars gene is repressed. Moreover, ars mRNA accumulation is dramatically stimulated by light, indicating that 1.4 kb of the csbp 5'-untranslated region are sufficient to confer light-dependent expression on the ars reporter gene.
Plant Mol Biol 1998 Apr
PMID:The Calvin cycle enzyme sedoheptulose-1,7-bisphosphatase is encoded by a light-regulated gene in Chlamydomonas reinhardtii. 952 Feb 83

Plastids contain a NAD(P)H-plastoquinone-oxidoreductase (NDH complex) which is homologous to the eubacterial and mitochondrial NADH-ubiquinone-oxidoreductase (complex I), but the metabolic function of the enzyme is unknown. The enzyme consists of at least eleven subunits (A-K), which are all encoded on the plastid chromosome. We have mutagenized ndhC and ndhJ by insertion, and ndhK and ndhA-I by deletion and insertion, of a cassette which carried a spectinomycin resistance gene as a marker. The transformation was carried out by the polyethylene glycol-mediated plastid transformation method. Southern analysis revealed that even after repeated regeneration cycles each of the four different types of transformants had retained 1-5% of wild-type gene copies. This suggests that complete deletion of ndh genes is not compatible with viability. The transformants displayed two characteristic phenotypes: (i) they lack the rapid rise in chlorophyll fluorescence in the dark after illumination with actinic light for 5 min; in the wild-type this dark-rise reflects a transient reduction of the plastoquinone pool by reduction equivalents generated in the stroma; and (ii) transformants with defects in the ndhC-K-J operon accumulate starch, indicating inefficient oxidation of glucose via glycolysis and the oxidative pentose phosphate pathway. Both observations support the theory of chlororespiration, which postulates that the NDH complex acts as a valve to remove excess reduction equivalents in the chloroplast.
Mol Gen Genet 1998 Apr
PMID:Mutagenesis of the genes encoding subunits A, C, H, I, J and K of the plastid NAD(P)H-plastoquinone-oxidoreductase in tobacco by polyethylene glycol-mediated plastome transformation. 961 85

When E. coli cells were grown in minimal medium supplemented with D-ribose and D-xylose, a diauxic growth preferring D-xylose was observed. Transcription of the ribose (rbs) operon was repressed in the presence of D-xylose, phenotypically similar to catabolite repression by D-glucose, although D-ribose did not affect transcription of the xylose (xyl) operon. Complementation analysis with xylR revealed that the repression of the rbs operon by D-xylose is exerted at the transcriptional level through XylR, suggesting a novel mechanism for catabolite repression. Furthermore, it was shown that L-arabinose reduced transcriptions of both xyl and rbs operons, whereas the arabinose operon was not affected by D-xylose or D-ribose, suggesting a priority mechanism for pentose utilization.
Mol Cells 1998 Jun 30
PMID:Priority of pentose utilization at the level of transcription: arabinose, xylose, and ribose operons. 966 69

The three-dimensional structure of 6-phosphogluconate dehydrogenase (6PGDH) from the parasitic protozoan Trypanosoma brucei has been solved at 2.8 A resolution. This pentose phosphate pathway enzyme is NADP-dependent; NADPH generated in the reaction protects against oxidative stress. The enzyme crystallises in the space-group P3121 with a dimer in the asymmetric unit and cell dimensions a=b=135.13 A, c=116.74 A, alpha=beta=90 degrees, gamma=120 degrees. The structure has refined to R=18.6% (Rfree=27.3%) with good geometry. The amino acid sequence of T. brucei 6PGDH is only 35% identical to that of the sheep liver enzyme and significant activity differences have been observed. The active dimer assembles with the C-terminal tail of one subunit threaded through the other, forming part of the substrate binding site. The tail of T. brucei 6PGDH is shorter than that of the sheep enzyme and its terminal residues associate tightly with the second monomer. The three-dimensional structure shows this generates additional interactions between the subunits close to the active site; the coenzyme binding domain is thereby associated more tightly with the helical domain. Three residues, conserved in all other known sequences, are important in creating a salt bridge between monomers close to the substrate binding site. The differences could explain the 200-fold enhanced affinity observed for the substrate analogue 6-phospho-2-deoxy-D-gluconate and suggest targets for anti-parasite drug design. The coenzyme binding domain of 6PGDH has a beta-alpha-beta fold; while in most species the "fingerprint" sequence is GxAxxG, in the T. brucei enzyme it is GxGxxG. Additional interactions between the enzyme and the coenzyme bis-phosphate are likely in the parasite 6PGDH, accounting for greater inhibition (40-fold) of 2'5'-ADP. While the core of the T. brucei dimer was restrained during refinement, several conformational differences have been found between the monomers; those at the coenzyme binding site suggest the molecule could be asymmetric during the enzyme reaction.
J Mol Biol 1998 Sep 25
PMID:A 2.8 A resolution structure of 6-phosphogluconate dehydrogenase from the protozoan parasite Trypanosoma brucei: comparison with the sheep enzyme accounts for differences in activity with coenzyme and substrate analogues. 973 29

ABC transport systems for import or export of nutrients and other substances across the cell membrane are widely distributed in nature. In most bacterial systems, a periplasmic component is the primary determinant of specificity of the transport complex as a whole. We report here the crystal structure of the periplasmic binding protein for the allose system (ALBP) from Escherichia coli, solved at 1.8 A resolution using the molecular replacement method. As in the other members of the family (especially the ribose binding protein, RBP, with which it shares 35 % sequence homology), this structure consists of two similar domains joined by a three-stranded hinge region. The protein is believed to exist in a dynamic equilibrium of closed and open conformations in solution which is an important part of its function. In the closed ligand-bound form observed here, D-allose is buried at the domain interface. Only the beta-anomer of allopyranose is seen in the crystal structure, although the alpha-anomer can potentially bind with a similar affinity. Details of the ligand-binding cleft reveal the features that determine substrate specificity. Extensive hydrogen bonding as well as hydrophobic interactions are found to be important. Altogether ten residues from both the domains form 14 hydrogen bonds with the sugar. In addition, three aromatic rings, one from each domain with faces parallel to the plane of the sugar ring and a third perpendicular, make up a hydrophobic stacking surface for the ring hydrogen atoms. Our results indicate that the aromatic rings forming the sugar binding cleft can sterically block the binding of any hexose epimer except D-allose, 6-deoxy-allose or 3-deoxy-glucose; the latter two are expected to bind with reduced affinity, due to the loss of some hydrogen bonds. The pyranose form of the pentose, D-ribose, can also fit into the ALBP binding cleft, although with lower binding affinity. Thus, ALBP can function as a low affinity transporter for D-ribose. The significance of these results is discussed in the context of the function of allose and ribose transport systems.
J Mol Biol 1999 Mar 12
PMID:Structure of D-allose binding protein from Escherichia coli bound to D-allose at 1.8 A resolution. 1006 13

Ribulose-5-phosphate 3-epimerase (EC 5.1.3.1) catalyzes the interconversion of ribulose-5-phosphate and xylulose-5-phosphate in the Calvin cycle and in the oxidative pentose phosphate pathway. The enzyme from potato chloroplasts was expressed in Escherichia coli, isolated and crystallized. The crystal structure was elucidated by multiple isomorphous replacement and refined at 2.3 A resolution. The enzyme is a homohexamer with D3 symmetry. The subunit chain fold is a (beta alpha)8-barrel. A sequence comparison with homologous epimerases outlined the active center and indicated that all members of this family are likely to share the same catalytic mechanism. The substrate could be modeled by putting its phosphate onto the observed sulfate position and its epimerized C3 atom between two carboxylates that participate in an extensive hydrogen bonding system. A mutation confirmed the crucial role of one of these carboxylates. The geometry together with the conservation pattern suggests that the negative charge of the putative cis-ene-diolate intermediate is stabilized by the transient induced dipoles of a methionine sulfur "cushion", which is proton-free and therefore prevents isomerization instead of epimerization.
J Mol Biol 1999 Apr 09
PMID:Structure and mechanism of the amphibolic enzyme D-ribulose-5-phosphate 3-epimerase from potato chloroplasts. 1019 Nov 44

Cell-fractionation and digitonin titration of procyclic trypomastigotes of Trypanosoma brucei, revealed that almost half of the total NADP+ -dependent glucose-6-phosphate dehydrogenase (G6PDH) activity, the first enzyme of the pentose phosphate pathway (PPP), is associated with glycosomes. The specific activity of G6PDH in the purified organelles was increased 4-fold relative to a total cell extract and showed latency. Moreover, in the absence of detergents this activity was totally resistant to the action of trypsin. The cytosolic counterpart was neither latent, nor was it resistant to trypsin. Both cytosolic and glycosomal G6PDH activities behaved identically on phenyl-, CM-, heparin-, and Affigel-blue-Sepharose columns. Both isoenzymes had a subunit Mr of 62 000 and an isoelectric point of 6.85, while kinetic studies carried out on the partially purified G6PDH from both cell compartments did not reveal any differences. The purified enzyme had an apparent Km of 138 and 5.3 microM for glucose 6-phosphate (G6P), and for NADP+, respectively, and had a specific activity of 14 micromol. (min mg of protein)(-1). We conclude that while in procyclic stages of T. brucei G6PDH activity is present in two different cell compartments, i.e. the cytosol and the glycosomes, these two activities most likely represent one and the same isoenzyme.
Mol Biochem Parasitol 1999 Mar 15
PMID:Purification, localisation and characterisation of glucose-6-phosphate dehydrogenase of Trypanosoma brucei. 1021 21

Chlamydia trachomatis is an obligate intracellular eubacteria that is dependent on a eukaryotic host cell for a variety of metabolites. For years, it has been speculated that chlamydiae are energy parasites, totally dependent on their host cell for ATP and other high-energy intermediates. To determine whether C. trachomatis contains functional enzymes that produce energy or reducing power, four enzymes involved in glycolysis or the pentose phosphate pathway, specifically pyruvate kinase, phosphoglycerate kinase, glyceraldehyde-3-phosphate dehydrogenase and glucose-6-phosphate dehydrogenase, were cloned, sequenced and expressed as recombinant proteins in Escherichia coli. The deduced amino acid sequences obtained show high homology to other pyruvate kinase, phosphoglycerate kinase, glyceraldehyde-3-phosphate dehydrogenase and glucose-6-phosphate dehydrogenase enzymes. In contrast to numerous other bacterial species, chlamydial glycolytic genes are not arranged in an operon, but are dispersed throughout the genome. Results from reverse transcriptase-polymerase chain reaction (RT-PCR) analysis indicate that all four genes are maximally expressed in the middle of the chlamydial developmental cycle. The chlamydial genes are capable of complementing mutant E. coli strains lacking the respective enzyme activities. In vitro enzyme analysis indicates that recombinant chlamydial enzymes expressed in E. coli are active and, interestingly, recombinant chlamydial pyruvate kinase is not regulated allosterically by fructose 1,6 bisphosphate or AMP, as found with other bacterial pyruvate kinases. In summary, identification and characterization of these glucose-catabolizing enzymes indicate that chlamydia contains the functional capacity to produce its own ATP and reducing power.
Mol Microbiol 1999 Jul
PMID:Glucose metabolism in Chlamydia trachomatis: the 'energy parasite' hypothesis revisited. 1041 34

In the light of recent findings on the effect of D-glucose upon D-fructose phosphorylation by human B-cell glucokinase, the influence of the aldohexose upon the metabolism of the ketohexose was investigated in rat pancreatic islets. D-glucose, although slightly decreasing D-[5-(3)H]fructose utilization, augmented the oxidation of the ketohexose, indicating that the aldohexose stimulates preferentially the oxidative, as distinct from anaerobic, modality of glycolysis. Such was not the case in parotid cells, taken as representative of functionally nonglucose-responsive cells. In the islets exposed to D-fructose, D-glucose also decreased the fractional contribution of the pentose shunt to the generation of CO2 and D-glyceraldehyde 3-phosphate from the ketohexose, and increased the inflow into the Krebs cycle of dicarboxylic metabolites relative to that of fructose-derived acetyl-CoA. This glucose-induced remodeling of D-fructose metabolism may optimize the insulin secretory response of islet cells to these hexoses, e.g. after food intake.
Mol Cell Biochem 1999 Jul
PMID:Hexose metabolism in pancreatic islets: effect of D-glucose upon D-fructose metabolism. 1048 41

To analyse genetic factors that potentially affect sugar quality and yield in Beta vulgaris, we designed primers based on 18 homologous ESTs and conserved regions of 32 heterologous ESTs encoding gene products that act in the Calvin cycle, the oxidative pentose phosphate cycle, photorespiration, synthesis, transport and degradation of sucrose, glycolysis, the citric acid cycle, nitrogen metabolism and osmoprotection. Data on the amplification of 54 gene homologues from B. vulgaris are presented. Among these are 35 homologues for which DNA sequence information from B. vulgaris is now available for the first time. For genetic mapping a PCR-based strategy using CAPS (cleaved amplified polymorphic sequence), DFLP (DNA fragment length polymorphism), SSCP (single-strand conformation polymorphism) and HD (heteroduplex) analysis was adopted. RFLP analysis was also used in some cases. The different techniques used for the detection of polymorphisms are evaluated with respect to their sensitivity and versatility. In all, 42 functional genes have been assigned to the nine linkage groups of sugar beet.
Mol Gen Genet 1999 Oct
PMID:PCR-based cloning and segregation analysis of functional gene homologues in Beta vulgaris. 1058 40


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