RESUMO
Trehalose-6-phosphate (T6P) is an intermediate of trehalose biosynthesis that plays an essential role in plant metabolism and development. Here, we comprehensively analyzed sequences from enzymes of trehalose metabolism in sugarcane, one of the main crops used for bioenergy production. We identified protein domains, phylogeny, and in silico expression levels for all classes of enzymes. However, post-translational modifications and residues involved in catalysis and substrate binding were analyzed only in trehalose-6-phosphate synthase (TPS) sequences. We retrieved 71 putative full-length TPS, 93 trehalose-6-phosphate phosphatase (TPP), and 3 trehalase (TRE) of sugarcane, showing all their conserved domains, respectively. Putative TPS (Classes I and II) and TPP sugarcane sequences were categorized into well-known groups reported in the literature. We measured the expression levels of the sequences from one sugarcane leaf transcriptomic dataset. Furthermore, TPS Class I has specific N-glycosylation sites inserted in conserved motifs and carries catalytic and binding residues in its TPS domain. Some of these residues are mutated in TPS Class II members, which implies loss of enzyme activity. Our approach retrieved many homo(eo)logous sequences for genes involved in trehalose metabolism, paving the way to discover the role of T6P signaling in sugarcane.
Assuntos
Saccharum , Trealose , Biologia Computacional , Glucosiltransferases/metabolismo , Poliploidia , Saccharum/genética , Saccharum/metabolismo , Trealase/genética , Trealose/genética , Trealose/metabolismoRESUMO
Trehalose-6-phosphate synthase OtsA from streptomycetes is unusual in that it uses GDP-glucose as the donor substrate rather than the more commonly used UDP-glucose. We now confirm that OtsA from Streptomyces venezuelae has such a preference for GDP-glucose and can utilize ADP-glucose to some extent too. A crystal structure of the enzyme shows that it shares twin Rossmann-like domains with the UDP-glucose-specific OtsA from Escherichia coli However, it is structurally more similar to Streptomyces hygroscopicus VldE, a GDP-valienol-dependent pseudoglycosyltransferase enzyme. Comparison of the donor binding sites reveals that the amino acids associated with the binding of diphosphoribose are almost all identical in these three enzymes. By contrast, the amino acids associated with binding guanine in VldE (Asn, Thr, and Val) are similar in S. venezuelae OtsA (Asp, Ser, and Phe, respectively) but not conserved in E. coli OtsA (His, Leu, and Asp, respectively), providing a rationale for the purine base specificity of S. venezuelae OtsA. To establish which donor is used in vivo, we generated an otsA null mutant in S. venezuelae The mutant had a cell density-dependent growth phenotype and accumulated galactose 1-phosphate, glucose 1-phosphate, and GDP-glucose when grown on galactose. To determine how the GDP-glucose is generated, we characterized three candidate GDP-glucose pyrophosphorylases. SVEN_3027 is a UDP-glucose pyrophosphorylase, SVEN_3972 is an unusual ITP-mannose pyrophosphorylase, and SVEN_2781 is a pyrophosphorylase that is capable of generating GDP-glucose as well as GDP-mannose. We have therefore established how S. venezuelae can make and utilize GDP-glucose in the biosynthesis of trehalose 6-phosphate.
Assuntos
Açúcares de Guanosina Difosfato/metabolismo , Streptomyces/metabolismo , Fosfatos Açúcares/biossíntese , Trealose/análogos & derivados , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Escherichia coli/genética , Escherichia coli/metabolismo , Galactose/genética , Galactose/metabolismo , Glucosiltransferases/genética , Glucosiltransferases/metabolismo , Açúcares de Guanosina Difosfato/genética , Streptomyces/genética , Fosfatos Açúcares/genética , Trealose/biossíntese , Trealose/genéticaRESUMO
Trehalose is a disaccharide with a wide range of applications in the food industry. We recently proposed a strategy for trehalose production based on a Corynebacterium glutamicum strain expressing the Escherichia coli enzyme UDP-glucose pyrophosphorylase (GalU). Biochemical network analysis suggest a further bottleneck for trehalose synthesis resulting from the coupling of phosphotransferase (PTS) mediated glucose uptake, and glucose catabolism in C. glutamicum. To overcome this coupling, we propose the expression of E. coli phosphoenolpyruvate synthetase (PpsA), in addition to GalU expression, in C. glutamicum. Although GalU expression improved trehalose synthesis in C. glutamicum, the simultaneous expression of GalU and PpsA did not result in a further increase in trehalose yield, but resulted in an increased catabolic rate of glucose, which could be ascribed to the operation of a futile cycle between phosphoenolpyruvate and pyruvate. The impact of GalU and PpsA expression on polysaccharide content, side product excretion and metabolic fluxes is discussed, as well as alternative ways to decouple glucose uptake and catabolism, in order to increase trehalose yield.
Assuntos
Corynebacterium glutamicum/metabolismo , Proteínas de Escherichia coli/biossíntese , Expressão Gênica/genética , Piruvato Sintase/biossíntese , Trealose/biossíntese , UTP-Glucose-1-Fosfato Uridililtransferase/biossíntese , Corynebacterium glutamicum/genética , Proteínas de Escherichia coli/genética , Engenharia Genética/métodos , Piruvato Sintase/genética , Trealose/genética , UTP-Glucose-1-Fosfato Uridililtransferase/genéticaRESUMO
Temperature shifts from 23 degrees C to 36 degrees C resulted in trehalose accumulation in Saccharomyces independently of genetic lesions in the cAMP-protein kinase cascade. In parallel, trehalose 6-phosphate synthase activity increased about 3-fold in all strains; the increase could be inhibited by cycloheximide, suggesting that protein synthesis was required. Heat shock treatment after the temperature shift led to a drastic increase in trehalose activity, and deactivation of the biosynthetic enzyme with a consequent drop in trehalose. Up to now no definite correlation between acquisition of thermotolerance and trehalose accumulation has been made.