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

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GlgE (EC is an a-maltose 1-phosphate:(1?4)-a-D-glucan 4-a-D-maltosyltransferase of the CAZy glycoside hydrolase 13_3 family. It is the defining enzyme of a bacterial a-glucan biosynthetic pathway and is a genetically validated anti-tuberculosis target. It catalyses the a-retaining transfer of maltosyl units from a-maltose 1-phosphate to maltooligosaccharides and is predicted to use a double displacement mechanism. Evidence for 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 a-maltose 1-phosphate bound to a D394A mutein and a ß-2-deoxy-2-fluoromaltosyl-enzyme intermediate with a E423A mutein were solved. There are few examples of CAZy glycoside hydrolase family 13 members that have had their glycosyl-enzyme intermediate structures solved 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.