title: 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.
authors.affiliation: Department of Biological Chemistry, John Innes Centre, Norwich Research Park , Norwich NR4 7UH, United Kingdom.
authors.forename: Karl
authors.initials: K
authors.lastname: Syson
authors.forename: Clare E M
authors.initials: CE
authors.lastname: Stevenson
authors.forename: Abdul M
authors.initials: AM
authors.lastname: Rashid
authors.forename: Gerhard
authors.initials: G
authors.lastname: Saalbach
authors.forename: Minhong
authors.initials: M
authors.lastname: Tang
authors.forename: Anne
authors.initials: A
authors.lastname: Tuukkanen
authors.forename: Dmitri I
authors.initials: DI
authors.lastname: Svergun
authors.forename: Stephen G
authors.initials: SG
authors.lastname: Withers
authors.forename: David M
authors.initials: DM
authors.lastname: Lawson
authors.forename: Stephen
authors.initials: S
authors.lastname: Bornemann
mesh_terms: D001483:Base Sequence; D018360:Crystallography, X-Ray; D017931:DNA Primers; D005964:Glucosyltransferases / Q000737:chemistry / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D012542:Scattering, Radiation; D048372:Streptomyces coelicolor / Q000201:enzymology*; D013379:Substrate Specificity; D013403:Sugar Phosphates / Q000378:metabolism*
chemical_list: D017931:DNA Primers; D013403:Sugar Phosphates; C080751:maltose 1-phosphate; D005964:Glucosyltransferases; C436710:maltosyltransferase