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.
Highlights
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
GDP-glucose Is the Preferred Donor Substrate of Recombinant S. venezuelae OtsA—The enzyme OtsA from S. hygroscopicus and other streptomycetes has been reported to have a preference for the donor GDP-glucose [15,16,17]
We have shown that S. venezuelae OtsA has a preference for GDP-glucose as the donor (Table 1), as has been reported for OtsA enzymes from other Streptomyces species [15,16,17] and other actinomycetes, such as Rubrobacter xylanophilus [18]
Summary
GDP-glucose Is the Preferred Donor Substrate of Recombinant S. venezuelae OtsA—The enzyme OtsA from S. hygroscopicus and other streptomycetes has been reported to have a preference for the donor GDP-glucose [15,16,17]. The Mutant Strain Accumulates GDP-glucose When Grown on Galactose—1H NMR spectroscopy of cell-free extracts of the otsA mutant strain grown in the presence of galactose revealed the presence of a number of metabolites (Fig. 5A) that were . Adding the cell-free extract to authentic galactose 1-phosphate and glucose 1-phosphate enhanced these specific resonances, consistent with these two metabolites accumulating in the mutant Another double doublet at 5.60 ppm (Fig. 5, A and C), together with additional resonances at low field, was suggestive of the presence of a nucleotide sugar diphosphate. With the absence of any known sequences of GDP-glucose pyrophosphorylases, we first identified three homologues of the GTP-dependent enzyme GDP-mannose pyrophosphorylase from M. tuberculosis (ManB Rv3264c) [32] in the genome of S. venezuelae We produced these three enzymes as recombinant proteins in E. coli. SVEN_3027 is a homologue of GalU from other organisms ( known as GtaB), which is
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