Limits...
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.

Show MeSH

Related in: MedlinePlus

Hydrogen-bonded water molecules near the 2-deoxy-2-fluoro-β-maltosylintermediate of the E423A mutein of GlgE (PDB entry 4CN4). The orientationshown is similar to that in Scheme 1 with subsites−1 and −2 labeled. Superimposed on the coordinates isthe final 2mFobs – DFcalc, αcalc electron density map calculatedat 2.4 Å resolution.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4048318&req=5

fig5: Hydrogen-bonded water molecules near the 2-deoxy-2-fluoro-β-maltosylintermediate of the E423A mutein of GlgE (PDB entry 4CN4). The orientationshown is similar to that in Scheme 1 with subsites−1 and −2 labeled. Superimposed on the coordinates isthe final 2mFobs – DFcalc, αcalc electron density map calculatedat 2.4 Å resolution.

Mentions: GlgE is efficientin catalyzing reversible phosphorolysis and transglycosidationreactions with little observable hydrolytic activity.1,10 The suppression of hydrolytic reactions could be due in part tothe binding of the acceptor affecting the conformation of the intermediate,as discussed above. In addition, inspection of the electron densitynear the intermediate revealed the presence of water molecules withinthe active site hydrogen-bonded to the 2′-OH group of the maltosylgroup and Tyr357 (Figure 5). Importantly, noneof these water molecules were positioned appropriately to attack C1of the intermediate, with the closest water molecule being 5.0 Åaway, and at an angle of ∼30° to the expected trajectory.This water molecule would have to move for the structure to accommodatean appropriately positioned water molecule for nucleophilic attack.Therefore, the structure is consistent with the possibility of GlgEutilizing this strategy to avoid hydrolysis, as has been suggestedfor other enzymes such as a GH31 family α-transglucosylase.22


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)

Hydrogen-bonded water molecules near the 2-deoxy-2-fluoro-β-maltosylintermediate of the E423A mutein of GlgE (PDB entry 4CN4). The orientationshown is similar to that in Scheme 1 with subsites−1 and −2 labeled. Superimposed on the coordinates isthe final 2mFobs – DFcalc, αcalc electron density map calculatedat 2.4 Å resolution.
© Copyright Policy
Related In: Results  -  Collection

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

fig5: Hydrogen-bonded water molecules near the 2-deoxy-2-fluoro-β-maltosylintermediate of the E423A mutein of GlgE (PDB entry 4CN4). The orientationshown is similar to that in Scheme 1 with subsites−1 and −2 labeled. Superimposed on the coordinates isthe final 2mFobs – DFcalc, αcalc electron density map calculatedat 2.4 Å resolution.
Mentions: GlgE is efficientin catalyzing reversible phosphorolysis and transglycosidationreactions with little observable hydrolytic activity.1,10 The suppression of hydrolytic reactions could be due in part tothe binding of the acceptor affecting the conformation of the intermediate,as discussed above. In addition, inspection of the electron densitynear the intermediate revealed the presence of water molecules withinthe active site hydrogen-bonded to the 2′-OH group of the maltosylgroup and Tyr357 (Figure 5). Importantly, noneof these water molecules were positioned appropriately to attack C1of the intermediate, with the closest water molecule being 5.0 Åaway, and at an angle of ∼30° to the expected trajectory.This water molecule would have to move for the structure to accommodatean appropriately positioned water molecule for nucleophilic attack.Therefore, the structure is consistent with the possibility of GlgEutilizing this strategy to avoid hydrolysis, as has been suggestedfor other enzymes such as a GH31 family α-transglucosylase.22

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