Nature of the multiple forms of sweet-potato glucose phosphate isomerase.
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ABSTRACT:
SUBMITTER: Phillips TL
PROVIDER: S-EPMC1165456 | biostudies-other | 1975 May
REPOSITORIES: biostudies-other
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Project description:Purple acid phosphatases (PAPs) are a family of binuclear metalloenzymes that catalyze the hydrolysis of phosphoric acid esters and anhydrides. A PAP in sweet potato has a unique, strongly antiferromagnetically coupled Fe(III)-Mn(II) center and is distinguished from other PAPs by its increased catalytic efficiency for a range of activated and unactivated phosphate esters, its strict requirement for Mn(II), and the presence of a mu-oxo bridge at pH 4.90. This enzyme displays maximum catalytic efficiency (k(cat)/K(m)) at pH 4.5, whereas its catalytic rate constant (k(cat)) is maximal at near-neutral pH, and, in contrast to other PAPs, its catalytic parameters are not dependent on the pK(a) of the leaving group. The crystal structure of the phosphate-bound Fe(III)-Mn(II) PAP has been determined to 2.5-A resolution (final R(free) value of 0.256). Structural comparisons of the active site of sweet potato, red kidney bean, and mammalian PAPs show several amino acid substitutions in the sweet potato enzyme that can account for its increased catalytic efficiency. The phosphate molecule binds in an unusual tripodal mode to the two metal ions, with two of the phosphate oxygen atoms binding to Fe(III) and Mn(II), a third oxygen atom bridging the two metal ions, and the fourth oxygen pointing toward the substrate binding pocket. This binding mode is unique among the known structures in this family but is reminiscent of phosphate binding to urease and of sulfate binding to lambda protein phosphatase. The structure and kinetics support the hypothesis that the bridging oxygen atom initiates hydrolysis.
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Project description:Sweet potato (Ipomoea batatas (L.) Lam.) is a good source of carbohydrates, an excellent raw material for starch-based industries, and a strong candidate for biofuel production due to its high starch content. However, the molecular basis of starch biosynthesis and accumulation in sweet potato is still insufficiently understood. Glucose-6-phosphate/phosphate translocators (GPTs) mediate the import of glucose-6-phosphate (Glc6P) into plastids for starch synthesis. Here, we report the isolation of a GPT-encoding gene, IbG6PPT1, from sweet potato and the identification of two additional IbG6PPT1 gene copies in the sweet potato genome. IbG6PPT1 encodes a chloroplast membrane-localized GPT belonging to the GPT1 group and highly expressed in storage root of sweet potato. Heterologous expression of IbG6PPT1 resulted in increased starch content in the leaves, root tips, and seeds and soluble sugar in seeds of Arabidopsis thaliana, but a reduction in soluble sugar in the leaves. These findings suggested that IbG6PPT1 might play a critical role in the distribution of carbon sources in source and sink and the accumulation of carbohydrates in storage tissues and would be a good candidate gene for controlling critical starch properties in sweet potato.
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Project description:RationaleMetabolic and structural remodeling is a hallmark of heart failure. This remodeling involves activation of the mTOR (mammalian target of rapamycin) signaling pathway, but little is known on how intermediary metabolites are integrated as metabolic signals.ObjectiveWe investigated the metabolic control of cardiac glycolysis and explored the potential of glucose 6-phosphate (G6P) to regulate glycolytic flux and mTOR activation.Methods and resultsWe developed a kinetic model of cardiomyocyte carbohydrate metabolism, CardioGlyco, to study the metabolic control of myocardial glycolysis and G6P levels. Metabolic control analysis revealed that G6P concentration is dependent on phosphoglucose isomerase (PGI) activity. Next, we integrated ex vivo tracer studies with mathematical simulations to test how changes in glucose supply and glycolytic flux affect mTOR activation. Nutrient deprivation promoted a tight coupling between glucose uptake and oxidation, G6P reduction, and increased protein-protein interaction between hexokinase II and mTOR. We validated the in silico modeling in cultured adult mouse ventricular cardiomyocytes by modulating PGI activity using erythrose 4-phosphate. Inhibition of glycolytic flux at the level of PGI caused G6P accumulation, which correlated with increased mTOR activation. Using click chemistry, we labeled newly synthesized proteins and confirmed that inhibition of PGI increases protein synthesis.ConclusionsThe reduction of PGI activity directly affects myocyte growth by regulating mTOR activation.
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Project description:To explore TNF-related genes in GPI-induced arthritis, we performed GeneChip analysis using arthritic splenocytes and control-immunized splenocytes. Among the arrayed TNFalpha-related genes, TIARP mRNA was highly expressed in arthritic splenocytes, with levels exceeding more than 20-times the control splenocytes
2009-07-24 | GSE17272 | GEO
Project description:To explore TNF-related genes in GPI-induced arthritis, we performed GeneChip analysis using arthritic splenocytes and control-immunized splenocytes. Among the arrayed TNFalpha-related genes, TIARP mRNA was highly expressed in arthritic splenocytes, with levels exceeding more than 20-times the control splenocytes The spleens of three GPI-GST (MW=89 kD) (300 µg) -immunized DBA/1 mice were harvested on day 10. As a control, the spleens of three GST (MW=26 kd) (100 µg) -immunized DBA/1 mice were used. Total RNA was extracted from the splenocytes using Isogen (Nippon gene), then 15 ug of RNA was utilized for cDNA synthesis by reverse transcription followed by synthesis of biotinylated cRNA through in vitro transcription. After cRNA fragmentation, hybridization with mouse 430A2.0 GeneChip (Affymetrix, Santa Clara, CA) with probes for 43,000 mouse genes ESTs was performed according to the protocol provided by the manufacturer. Analysis was performed by gene expression software
2010-03-03 | E-GEOD-17272 | biostudies-arrayexpress
Project description:The mechanism of implantation in carnivores is poorly understood. However, a previously unidentified 60-kDa protein has been shown to be necessary for embryo implantation in ferrets. Here we identify this protein as glucose-6-phosphate isomerase (GPI). GPI is expressed by the corpus luteum on days 6-9 of pregnancy, the time at which implantation-promoting activity has been found in corpora lutea. Passive immunization against GPI reduced the number of implantation sites in pregnant ferrets in a dose-dependent manner. GPI is a multifunctional protein. Although first identified for its role in glycolysis, GPI has since been implicated in neural growth, lymphocyte maturation, and metastasis. This study demonstrates a previously uncharacterized function of this protein that may represent the natural motility-stimulating activity that has been co-opted by tumor cells.
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Project description:Glucose content and in vitro bioaccessibility were determined in raw and cooked pulp of Arapey, Cuabé, and Beauregard sweet potato varieties, as well as Maravilla del Mercado and Atlas winter squash, after zero, two, four, and six months of storage (14 °C, 80% relative humidity (RH)). The total glucose content in 100 g of raw pulp was, for Arapey, 17.7 g; Beauregard, 13.2 g; Cuabé, 12.6 g; Atlas, 4.0 g; and in Maravilla del Mercado, 4.1 g. These contents were reduced by cooking process and storage time, 1.1 to 1.5 times, respectively, depending on the sweet potato variety. In winter squash varieties, the total glucose content was not modified by cooking, while the storage increased glucose content 2.8 times in the second month. After in vitro digestion, the glucose content released was 7.0 times higher in sweet potato (6.4 g) than in winter squash (0.91 g) varieties. Glucose released by in vitro digestion for sweet potato stored for six months did not change, but in winter squashes, stored Atlas released glucose content increased 1.6 times. In conclusion, in sweet potato and winter squash, the glucose content and the released glucose during digestive simulation depends on the variety and the storage time. These factors strongly affect the supply of glucose for human nutrition and should be taken into account for adjusting a diet according to consumer needs.
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Project description:Glycosylation contributes to the diversity and stability of anthocyanins in plants. The process is catalysed by various glucosyltransferases using different anthocyanidin aglycones and glycosyl donors. In this study, we found that an anthocyanidin 3-O-glucoside-2″-O-glucosyltransferase (3GGT) from purple sweet potato (Ipomoea batatas) catalyses the conversion of anthocyanidin 3-O-glucoside into anthocyanidin 3-O-sophoroside, which is functionally different from the 3GGT ortholog of Arabidopsis. Phylogenetic analysis indicated regioselectivity of 3GGT using uridine-5'-diphosphate (UDP)-xylose or UDP-glucose as the glycosyl is divergent between Convolvulaceae and Arabidopsis. Homology-based protein modeling and site-directed mutagenesis of Ib3GGT and At3GGT suggested that the Thr-138 of Ib3GGT is a key amino acid residue for UDP-glucose recognition and that it plays a major role in sugar-donor selectivity. Wild-type and ugt79b1 mutants (defective in UDP carbohydrate-dependent glycosyltransferases, UGTs) of Arabidopsis plants overexpressing Ib3GGT produced the new component cyanidin 3-O-sophoroside. Moreover, Ib3GGT expression was associated with anthocyanin accumulation in different tissues during I. batatas plant development and was regulated by the transcription factor IbMYB1. Localization assays for Ib3GGT showed that glycosyl extension occurs in the cytosol and not in the endoplasmic reticulum. This study therefore reveals the function of Ib3GGT in glycosyl extension of anthocyanins and demonstrates that Thr-138 is the key amino acid residue for UDP-glucose recognition.
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