title: The Production and Utilization of GDP-glucose in the Biosynthesis of Trehalose 6-Phosphate by Streptomyces venezuelae.
abstract: 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.
authors.affiliation: the Laboratorio de Enzimología Molecular, Instituto de Agrobiotecnología del Litoral (UNL-CONICET), Facultad de Bioquímica y Ciencias Biológicas, CCT-Santa Fe, Colectora Ruta Nac 168 Km 0, 3000 Santa Fe, Argentina.
authors.forename: Matías D
authors.initials: MD
authors.lastname: Asención Diez
authors.affiliation: From the Biological Chemistry Department, John Innes Centre, Norwich Research Park, Norwich NR4 7UH, United Kingdom and.
authors.forename: Farzana
authors.initials: F
authors.lastname: Miah
authors.affiliation: From the Biological Chemistry Department, John Innes Centre, Norwich Research Park, Norwich NR4 7UH, United Kingdom and.
authors.forename: Clare E M
authors.initials: CE
authors.lastname: Stevenson
authors.affiliation: From the Biological Chemistry Department, John Innes Centre, Norwich Research Park, Norwich NR4 7UH, United Kingdom and.
authors.forename: David M
authors.initials: DM
authors.lastname: Lawson
authors.affiliation: the Laboratorio de Enzimología Molecular, Instituto de Agrobiotecnología del Litoral (UNL-CONICET), Facultad de Bioquímica y Ciencias Biológicas, CCT-Santa Fe, Colectora Ruta Nac 168 Km 0, 3000 Santa Fe, Argentina.
authors.forename: Alberto A
authors.initials: AA
authors.lastname: Iglesias
authors.affiliation: From the Biological Chemistry Department, John Innes Centre, Norwich Research Park, Norwich NR4 7UH, United Kingdom and stephen.bornemann@jic.ac.uk.
authors.forename: Stephen
authors.initials: S
authors.lastname: Bornemann
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D001665:Binding Sites; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005690:Galactose / Q000235:genetics / Q000378:metabolism; D005964:Glucosyltransferases / Q000235:genetics / Q000378:metabolism; D006156:Guanosine Diphosphate Sugars / Q000235:genetics / Q000378:metabolism*; D013302:Streptomyces / Q000235:genetics / Q000378:metabolism*; D013403:Sugar Phosphates / Q000096:biosynthesis* / Q000235:genetics; D014199:Trehalose / Q000031:analogs & derivatives* / Q000096:biosynthesis / Q000235:genetics
chemical_list: D001426:Bacterial Proteins; C480160:GDP-gulose; D006156:Guanosine Diphosphate Sugars; D013403:Sugar Phosphates; C082722:trehalose-6-phosphate; D014199:Trehalose; D005964:Glucosyltransferases; C023180:trehalose-6-phosphate synthase; D005690:Galactose
keywords: carbohydrate metabolism; crystal structure; glycosyltransferase; microbiology; substrate specificity; trehalose