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

Abstract

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 Streptomyces coelicolor 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 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.

α-Glucan biosynthesis and the GlgE pathway in Mycobacterium tuberculosis

“…This would appear to increase flux from G1P through to ADPG and favour flux through to GlgA. Another glycolytic intermediate, [46]. These structures are consistent with the double displacement mechanism shown.…”

“…In order to obtain a structure with M1P bound, it was necessary to substitute the Asp 394 side chain with alanine to prevent the slow hydrolysis of this substrate [46]. This confirmed the location of the donor-binding site to be adjacent to the predicted catalytic residues (Figure 2).…”

“…The authors of this study suggest that the difluoro substitution destablizes a putative oxocarbenium transition state through electron withdrawal to such an extent that the fluorine at the anomeric position is too poor a leaving group to allow the first catalytic step to occur. Importantly, the prediction that the S. coelicolor and M. tuberculosis enzymes have very similar donor sites based on very similar kinetic properties [17,33,46] has now been confirmed [48]. This will no doubt help inform drug design.…”

Diversity of phosphorylases in glycoside hydrolase families

“…This is the only phosphorylase that is considered to be involved in the synthesis of glycoside. After the publication of this report, a series of reports considering the inhibitor of GlgE were published with the expectation that they may aid the development of a novel drug for multidrug-resistant strains of M. tuberculosis, the notorious pathogenic bacterium that causes tuberculosis (Kalscheuer et al 2010;Leiba et al 2013;Veleti et al 2014;Syson et al 2014;Sengupta et al 2014Sengupta et al , 2015. The 3D structure of the enzyme from Streptomyces coelicolor is available (Syson et al 2011).…”

Synthesis of a Poly-hydroxypyrolidine-Based inhibitor of Mycobacterium tuberculosis GlgE

“…7 Recently, Syson et al trapped Sco GlgE with 2-deoxy-2-fluoro-α-maltosyl fluoride, which provided additional evidence for a double-displacement mechanism proceeding through a transition state as shown in Scheme 1. 8 The first step of the reaction is nucleophilic attack of Asp418 on the anomeric center of M1P to generate a β-maltosyl enzyme intermediate. The enzyme intermediate is attacked by a glucan to extend the chain.…”