Structural Insight into How Streptomyces coelicolor Maltosyl Transferase GlgE Binds alpha-Maltose 1-Phosphate and Forms a Maltosyl-enzyme Intermediate

Karl Syson, Clare E. M. Stevenson, Abdul M. Rashid, Gerhard Saalbach, Minhong Tang, Anne Tuukkanen, Dmitri I. Svergun, Stephen G. Withers, David M. Lawson, Stephen Bornemann

Research output: Contribution to journalArticlepeer-review

24 Citations (Scopus)

Abstract

GlgE (EC 2.4.99.16) is an alpha-maltose 1-phosphate:(1 -> 4)-alpha-D-glucan 4-alpha-D-maltosyltransferase of the CAZy glycoside hydrolase 13_3 family. It is the defining enzyme of a bacterial alpha-glucan biosynthetic pathway and is a genetically validated anti-tuberculosis target. It catalyzes the alpha-retaining transfer of maltosyl units from alpha-maltose 1-phosphate to maltooligosaccharides and is predicted to use a double-displacement mechanism. Evidence of this mechanism was obtained using a combination of site-directed mutagenesis of Streptomyces coelicolor GlgE isoform I, substrate analogues, protein crystallography, and mass spectrometry. The X-ray structures of alpha-maltose 1-phosphate bound to a D394A mutein and a beta-2-deoxy-2-fluoromaltosyl-enzyme intermediate with a E423A mutein were determined. There are few examples of CAZy glycoside hydrolase family 13 members that have had their glycosyl-enzyme intermediate structures determined, and none before now have been obtained with a 2-deoxy-2-fluoro substrate analogue. The covalent modification of Asp394 was confirmed using mass spectrometry. A similar modification of wild-type GlgE proteins from S. coelicolor and Mycobacterium tuberculosis was also observed. Small-angle X-ray scattering of the M. tuberculosis enzyme revealed a homodimeric assembly similar to that of the S. coelicolor enzyme but with slightly differently oriented monomers. The deeper understanding of the structure function relationships of S. coelicolor GlgE will aid the development of inhibitors of the M. tuberculosis enzyme.

Original languageEnglish
Pages (from-to)2494-2504
Number of pages11
JournalBiochemistry
Volume53
Issue number15
Early online date11 Apr 2014
DOIs
Publication statusPublished - 22 Apr 2014
Externally publishedYes

Keywords

  • VALIDATED ANTITUBERCULOSIS TARGET
  • HYDROLASE FAMILY 31
  • MYCOBACTERIUM-TUBERCULOSIS
  • CRYSTAL-STRUCTURE
  • MALTOSYLTRANSFERASE GLGE
  • GLYCOSIDASE INHIBITORS
  • CATALYTIC MECHANISM
  • SOLUTION SCATTERING
  • MOLECULAR-GRAPHICS
  • AMYLASE FAMILY

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