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

Syson K, Stevenson CE, Rashid AM, Saalbach G, Tang M, Tuukkanen A, Svergun DI, Withers SG, Lawson DM, Bornemann S - Biochemistry (2014)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Chemistry, John Innes Centre, Norwich Research Park , Norwich NR4 7UH, United Kingdom.

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.

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Domains within a dimerof S. coelicolor GlgE isoformI (PDB entry 3ZSS).10
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fig1: Domains within a dimerof S. coelicolor GlgE isoformI (PDB entry 3ZSS).10

Mentions: We havepreviously reported structures of S. coelicolor GlgEisoform I (PDB entry 3ZSS in Figure 1 and Figure S1A of the Supporting Information), including those withα-maltose bound (PDB entry 3ZT5 in Figure S1B of the Supporting Information).10 Theputative nucleophile (Asp394) and general acid/base (Glu423) catalyticresidues were close to the reducing end of the maltose, suggestingthe glucose rings occupied subsites −1 and −242 of the donor substrate, α-maltose 1-phosphate.However, it was not possible to obtain a structure of GlgE with thedonor bound, presumably because it was slowly but excessively hydrolyzedover the time scale of protein crystallization.


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

Syson K, Stevenson CE, Rashid AM, Saalbach G, Tang M, Tuukkanen A, Svergun DI, Withers SG, Lawson DM, Bornemann S - Biochemistry (2014)

Domains within a dimerof S. coelicolor GlgE isoformI (PDB entry 3ZSS).10
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4048318&req=5

fig1: Domains within a dimerof S. coelicolor GlgE isoformI (PDB entry 3ZSS).10
Mentions: We havepreviously reported structures of S. coelicolor GlgEisoform I (PDB entry 3ZSS in Figure 1 and Figure S1A of the Supporting Information), including those withα-maltose bound (PDB entry 3ZT5 in Figure S1B of the Supporting Information).10 Theputative nucleophile (Asp394) and general acid/base (Glu423) catalyticresidues were close to the reducing end of the maltose, suggestingthe glucose rings occupied subsites −1 and −242 of the donor substrate, α-maltose 1-phosphate.However, it was not possible to obtain a structure of GlgE with thedonor bound, presumably because it was slowly but excessively hydrolyzedover the time scale of protein crystallization.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Chemistry, John Innes Centre, Norwich Research Park , Norwich NR4 7UH, United Kingdom.

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.

Show MeSH
Related in: MedlinePlus