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The production and utilization of GDP-glucose in the biosynthesis of trehalose 6-phosphate by Streptomyces venezuelae

DOI: 10.1074/jbc.M116.758664 DOI Help

Authors: Matías D. Asención Diez (Instituto de Agrobiotecnología del Litoral (UNL-CONICET)) , Farzana Miah (John Innes Centre) , Clare E. M. Stevenson (John Innes Centre) , David M. Lawson (John Innes Centre) , Alberto A. Iglesias (Instituto de Agrobiotecnología del Litoral (UNL-CONICET)) , Stephen Bornemann (John Innes Centre)
Co-authored by industrial partner: No

Type: Journal Paper
Journal: Journal Of Biological Chemistry , VOL 292 , PAGES 945 - 954

State: Published (Approved)
Published: January 2017
Diamond Proposal Number(s): 7641

Open Access Open Access

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 utilise 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 pseudo-glycosyltransferase enzyme. Comparison of the donor binding sites reveals that the amino acids associated with the binding of diphosphoribose are almost completely conserved between these three enzymes. By contrast, the amino acids associated with binding guanine in VldE (Asn, Thr and Val) are functionally conserved in S. venezuelae OtsA (Asp, Ser and Phe, respectively) but not 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. To determine how the GDP-glucose is generated, we characterised 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 utilise GDP-glucose in the biosynthesis of trehalose-6-phosphate.

Journal Keywords: trehalose; glycosyltransferase; crystal structure; carbohydrate metabolism; microbiology; substrate specificity

Diamond Keywords: Bacteria; Enzymes

Subject Areas: Biology and Bio-materials, Chemistry

Diamond Offline Facilities: No
Instruments: I04-1-Macromolecular Crystallography (fixed wavelength)

Added On: 09/03/2017 09:28

Documents:
945.full.pdf

Discipline Tags:

Biochemistry Chemistry Structural biology Life Sciences & Biotech

Technical Tags:

Diffraction Macromolecular Crystallography (MX)