RESUMO
Sugarcane is a monocot plant that accumulates sucrose to levels of up to 50% of dry weight in the stalk. The mechanisms that are involved in sucrose accumulation in sugarcane are not well understood, and little is known with regard to factors that control the extent of sucrose storage in the stalks. UDP-glucose pyrophosphorylase (UGPase; EC 2.7.7.9) is an enzyme that produces UDP-glucose, a key precursor for sucrose metabolism and cell wall biosynthesis. The objective of this work was to gain insights into the ScUGPase-1 expression pattern and regulatory mechanisms that control protein activity. ScUGPase-1 expression was negatively correlated with the sucrose content in the internodes during development, and only slight differences in the expression patterns were observed between two cultivars that differ in sucrose content. The intracellular localization of ScUGPase-1 indicated partial membrane association of this soluble protein in both the leaves and internodes. Using a phospho-specific antibody, we observed that ScUGPase-1 was phosphorylated in vivo at the Ser-419 site in the soluble and membrane fractions from the leaves but not from the internodes. The purified recombinant enzyme was kinetically characterized in the direction of UDP-glucose formation, and the enzyme activity was affected by redox modification. Preincubation with H2O2 strongly inhibited this activity, which could be reversed by DTT. Small angle x-ray scattering analysis indicated that the dimer interface is located at the C terminus and provided the first structural model of the dimer of sugarcane UGPase in solution.
Assuntos
Membrana Celular/enzimologia , Regulação Enzimológica da Expressão Gênica/fisiologia , Regulação da Expressão Gênica de Plantas/fisiologia , Proteínas de Plantas/biossíntese , Caules de Planta/enzimologia , Saccharum/enzimologia , UTP-Glucose-1-Fosfato Uridililtransferase/biossíntese , Membrana Celular/química , Modelos Moleculares , Fosforilação/fisiologia , Proteínas de Plantas/química , Caules de Planta/química , Estrutura Terciária de Proteína , UTP-Glucose-1-Fosfato Uridililtransferase/química , Uridina Difosfato Glucose/biossíntese , Uridina Difosfato Glucose/químicaRESUMO
A cluster of five genes, proposed to be involved in the formation of extracellular polysaccharide (EPS) precursors via the Leloir pathway, have been identified in the acidophilic autotroph Acidithiobacillus ferrooxidans. The order of the genes is luxA-galE-galK-pgm-galM, encoding a LuxA-like protein, UDP-glucose 4-epimerase, galactokinase, phosphoglucomutase, and galactose mutarotase, respectively. The gal cluster forms a single transcriptional unit and is therefore an operon. Two other putative genes of the Leloir pathway, galU, potentially encoding UDP-glucose pyrophosphorylase, and a gene designated galT-like, which may encode a galactose-1-phosphate uridylyltransferase-like activity, were found unlinked in the genome. Using semiquantitative reverse transcription-PCR, the genes of the gal operon were shown to be expressed more during growth in iron medium than in growth in sulfur medium. The functions of galE, pgm, galU, and the galT-like gene were validated by complementation of Escherichia coli mutants and by in vitro enzyme assays. The data suggest that A. ferrooxidans is capable of synthesizing the EPS precursors UDP-glucose and UDP-galactose. In addition, genes rfbA, -B, -C, and -D were identified in the genome of A. ferrooxidans, suggesting that it can also synthesize the EPS precursor dTDP-rhamnose. Since EPSs constitute the major bulk of biofilms, this study may provide an initial model for the metabolic pathways involved in biofilm formation in A. ferrooxidans and aid in understanding the role of biofilms in mineral leaching and the formation of acid mine drainage.
Assuntos
Acidithiobacillus/metabolismo , Proteínas de Bactérias/metabolismo , Família Multigênica , Polissacarídeos Bacterianos/biossíntese , Uridina Difosfato Galactose/biossíntese , Uridina Difosfato Glucose/biossíntese , Acidithiobacillus/enzimologia , Acidithiobacillus/genética , Acidithiobacillus/crescimento & desenvolvimento , Proteínas de Bactérias/genética , Carboidratos Epimerases/genética , Carboidratos Epimerases/metabolismo , Galactoquinase/genética , Galactoquinase/metabolismo , Dados de Sequência Molecular , Fosfoglucomutase/genética , Fosfoglucomutase/metabolismo , Análise de Sequência de DNA , UDPglucose 4-Epimerase/genética , UDPglucose 4-Epimerase/metabolismoRESUMO
The synthesis of periplasmic beta(1-2)glucan is required for crown gall tumor formation by Agrobacterium tumefaciens and for effective nodulation of alfalfa by Rhizobium meliloti. The exoC (pscA) gene is required for this synthesis by both bacteria as well as for the synthesis of capsular polysaccharide and normal lipopolysaccharide. We tested the possibility that the pleiotropic ExoC phenotype is due to a defect in the synthesis of an intermediate common to several polysaccharide biosynthetic pathways. Cytoplasmic extracts from wild-type A. tumefaciens and from exoC mutants of A. tumefaciens containing a cloned wild-type exoC gene synthesized in vitro UDP-glucose from glucose, glucose 1-phosphate, and glucose 6-phosphate. Extracts from exoC mutants synthesized UDP-glucose from glucose 1-phosphate but not from glucose or glucose 6-phosphate. Membranes from exoC mutant cells synthesized beta(1-2)glucan in vitro when exogenous UDP-glucose was added and contained the 235-kilodalton protein, which has been shown to carry out this synthesis in wild-type cells. We conclude that the inability of exoC mutants to synthesize beta(1-2)glucan is due to a deficiency in the activity of the enzyme phosphoglucomutase (EC 2.7.5.1), which in wild-type bacteria converts glucose 6-phosphate to glucose 1-phosphate, an intermediate in the synthesis of UDP-glucose. This interpretation can account for all of the deficiencies in polysaccharide synthesis which have been observed in these mutants.