Structural Insight into How <i>Streptomyces coelicolor</i> Maltosyl Transferase GlgE Binds α‑Maltose 1‑Phosphate and Forms a Maltosyl-enzyme Intermediate

GlgE (EC 2.4.99.16) is an α-maltose 1-phosphate:(1→4)-α-d-glucan 4-α-d-maltosyltransferase of the CAZy glycoside hydrolase 13_3 family. It is the defining enzyme of a bacterial α-glucan biosynthetic pathway and is a genetically validated anti-tuberculosis target. It catalyzes the α-retaining transfer of maltosyl units from α-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 <i>Streptomyces coelicolor</i> GlgE isoform I, substrate analogues, protein crystallography, and mass spectrometry. The X-ray structures of α-maltose 1-phosphate bound to a D394A mutein and a β-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 <i>S. coelicolor</i> and <i>Mycobacterium tuberculosis</i> was also observed. Small-angle X-ray scattering of the <i>M. tuberculosis</i> enzyme revealed a homodimeric assembly similar to that of the <i>S. coelicolor</i> enzyme but with slightly differently oriented monomers. The deeper understanding of the structure–function relationships of <i>S. coelicolor</i> GlgE will aid the development of inhibitors of the <i>M. tuberculosis</i> enzyme