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.

Glucosamine-6P and glucosamine-1P, respectively an activator and a substrate of rhodococcal ADP-glucose pyrophosphorylases, show a hint to ascertain (actino)bacterial glucosamine metabolism

“…In this context, the ADP-GlcN synthesis inside rhodococci cells could be inferred, although this framework needs of future experimental studies (actually, ongoing experiments in our group). Previously, we demonstrated that a new type of nucleotide-sugar pyrophosphorylase (GDP-GlcPPase, EC 2.7.7.34) from Streptomyces venezuelae (a close related organism to rhodococci) produced metabolically significant amounts of the specific glucosyl donor (GDP-Glc) for trehalose synthesis [17]. This new enzyme showed an efficiency toward GDP-Glc synthesis in the order of 10 −1 s -1 mM -1 , similar to parameters presented in this work for both rhodococcal ADP-GlcPPases sustaining our proposed scenario.…”

Mycobacterial OtsA Structures Unveil Substrate Preference Mechanism and Allosteric Regulation by 2-Oxoglutarate and 2-Phosphoglycerate

“…Data collection and refinement statistics are summarized in Table S1. Mtr OtsA is composed of two Rossmann-fold domains with a deep catalytic site at their interface, in an arrangement typical of GT-B glycosyltransferases as described previously for other organisms (27, 29, 30). The apo protein crystallized in the I4 1 22 space group, with one protomer per asymmetric unit, and diffracted to ∼1.8-Å resolution.…”

“…Indeed, in E. coli both tetrameric and dimeric forms were reported (27) while in Streptomyces venezuelae and Aspergillus fumigatus only the dimeric form was observed (29, 30). However, in mycobacteria and closely related organisms the tetramer interfaces are highly conserved, suggesting a tetrameric assembly of OtsA in all of these organisms (Fig.…”

“…OtsAs from different organisms have been shown to have different donor substrate preferences, while being capable of using several nucleotide donors (30, 33, 36, 37), which is reflected in lower conservation of the donor substrate-interacting residues (Fig. S2).…”

“…Binding of the substrates occurs sequentially in a “bi-bi mechanism” with NDP-glucose binding first and G6P second (26, 28). Interestingly, OtsAs from different organisms show different donor preferences as shown by kinetic studies and X-ray crystal structures, but the reasons behind these preferences are poorly understood (27, 29–33). Recently, different roles were identified for OtsA beyond its enzymatic activity.…”