Structural Insight into How Streptomyces coelicolor Maltosyl Transferase GlgE Binds $\alpha$-Maltose 1-Phosphate and Forms a Maltosyl-Enzyme Intermediate

Syson, Karl; Tuukkanen, Anne; Lawson, David M.; Stevenson, Clare E. M.; Svergun, Dmitri; Saalbach, Gerhard; Withers, Stephen G.; Tang, Minhong; Rashid, Abdul M.; Bornemann, Stephen

doi:10.1021/bi500183c

Journal Article

PUBDB-2015-01352

Structural Insight into How Streptomyces coelicolor Maltosyl Transferase GlgE Binds $\alpha$-Maltose 1-Phosphate and Forms a Maltosyl-Enzyme Intermediate

Syson, K. (Corresponding Author) ; Stevenson, C. E. M. ; Rashid, A. M. ; Saalbach, G. ; Tang, M. ; Tuukkanen, A.>Extern* ; Svergun, D.>Extern*EMBL* ; Withers, S. G. ; Lawson, D. M. ; Bornemann, S.

2014
American Chemical Society Columbus, Ohio

Biochemistry 53(15), 2494 - 2504 (2014) [10.1021/bi500183c]

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Please use a persistent id in citations: doi:10.1021/bi500183c

Abstract: GlgE (EC 2.4.99.16) is an α-maltose 1-phosphate:(1→4)-α- D -glucan 4-α- D -maltosyltransferase of theCAZy glycoside hydrolase 13_3 family. It is the definingenzyme of a bacterial α-glucan biosynthetic pathway and is agenetically 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 adouble-displacement mechanism. Evidence of this mechanismwas 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 aβ-2-deoxy-2-fluoromaltosyl-enzyme intermediate with a E423A mutein were determined. There are few examples of CAZyglycoside hydrolase family 13 members that have had their glycosyl-enzyme intermediate structures determined, and none beforenow have been obtained with a 2-deoxy-2-fluoro substrate analogue. The covalent modification of Asp394 was confirmed usingmass spectrometry. A similar modification of wild-type GlgE proteins from S. coelicolor and Mycobacterium tuberculosis was alsoobserved. 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−functionrelationships of S. coelicolor GlgE will aid the development of inhibitors of the M. tuberculosis enzyme.

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