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Ligand-bound structures and site-directed mutagenesis identify the acceptor and secondary binding sites of streptomyces coelicolor maltosyltransferase GlgE*

DOI: 10.1074/jbc.M116.748160 DOI Help

Authors: Karl Syson (John Innes Centre) , Clare E. M. Stevenson (John Innes Centre) , Farzana Miah (John Innes Centre) , J. Elaine Barclay (John Innes Centre) , Minhong Tang (John Innes Centre) , Andrii Gorelik (John Innes Centre) , Abdul M. Rashid (John Innes Centre) , David M. Lawson (John Innes Centre) , Stephen Bornemann (John Innes Centre)
Co-authored by industrial partner: No

Type: Journal Paper
Journal: Journal Of Biological Chemistry , VOL 291 , PAGES 21531 - 21540

State: Published (Approved)
Published: October 2016
Diamond Proposal Number(s): 1219 , 9475

Open Access Open Access

Abstract: GlgE is a maltosyltransferase involved in α-glucan biosynthesis in bacteria that has been genetically validated as a target for tuberculosis therapies. Crystals of the Mycobacterium tuberculosis enzyme diffract at low resolution so most structural studies have been with the very similar Streptomyces coelicolor GlgE isoform 1. Although the donor binding site for α-maltose 1-phosphate had been previously structurally defined, the acceptor site had not. Using mutagenesis, kinetics, and protein crystallography of the S. coelicolor enzyme, we have now identified the +1 to +6 subsites of the acceptor/product, which overlap with the known cyclodextrin binding site. The sugar residues in the acceptor subsites +1 to +5 are oriented such that they disfavor the binding of malto-oligosaccharides that bear branches at their 6-positions, consistent with the known acceptor chain specificity of GlgE. A secondary binding site remote from the catalytic center was identified that is distinct from one reported for the M. tuberculosis enzyme. This new site is capable of binding a branched α-glucan and is most likely involved in guiding acceptors toward the donor site because its disruption kinetically compromises the ability of GlgE to extend polymeric substrates. However, disruption of this site, which is conserved in the Streptomyces venezuelae GlgE enzyme, did not affect the growth of S. venezuelae or the structure of the polymeric product. The acceptor subsites +1 to +4 in the S. coelicolor enzyme are well conserved in the M. tuberculosis enzyme so their identification could help inform the design of inhibitors with therapeutic potential.

Journal Keywords: carbohydrate-binding protein; crystal structure; glycosyltransferase; oligosaccharide; site-directed mutagenesis

Diamond Keywords: Tuberculosis (TB); Bacteria; Enzymes

Subject Areas: Biology and Bio-materials, Chemistry, Medicine

Instruments: I04-1-Macromolecular Crystallography (fixed wavelength) , I24-Microfocus Macromolecular Crystallography

Added On: 17/11/2016 13:25


Discipline Tags:

Pathogens Infectious Diseases Health & Wellbeing Biochemistry Chemistry Structural biology Drug Discovery Life Sciences & Biotech

Technical Tags:

Diffraction Macromolecular Crystallography (MX)