Structural insight into how Streptomyces coelicolor maltosyl transferase GlgE binds ?-maltose 1-phosphate and forms a maltosyl-enzyme intermediate.

ABSTRACT: GlgE (EC 2.4.99.16) is an ?-maltose 1-phosphate:(1?4)-?-d-glucan 4-?-d-maltosyltransferase of the CAZy glycoside hydrolase 13_3 family. It is the defining enzyme of a bacterial ?-glucan biosynthetic pathway and is a genetically validated anti-tuberculosis target. It catalyzes the ?-retaining transfer of maltosyl units from ?-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 ?-maltose 1-phosphate bound to a D394A mutein and a ?-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.

SUBMITTER: Syson K 

PROVIDER: S-EPMC4048318 | biostudies-literature | 2014 Apr

REPOSITORIES: biostudies-literature