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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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Structures of GlgE with (A) α-maltose 1-phosphate bound tothe D394A mutein (PDB entry 4CN1) and (B) the covalent intermediate formed betweenthe E423A mutein and 2-deoxy-2-fluoro-α-maltosyl fluoride (PDBentry 4CN4).Difference electron density “omit” maps were generatedfor bound ligands using phases from final models without ligand coordinatesafter application of small random shifts to the models and re-refining.The corresponding stereo images are shown in Figure S1 of the Supporting Information. Some amino acids interactingwith the ligands have been omitted for the sake of clarity, but allare shown in Figure S2 of the Supporting Information. Subsites −1 and −2 are labeled.
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fig2: Structures of GlgE with (A) α-maltose 1-phosphate bound tothe D394A mutein (PDB entry 4CN1) and (B) the covalent intermediate formed betweenthe E423A mutein and 2-deoxy-2-fluoro-α-maltosyl fluoride (PDBentry 4CN4).Difference electron density “omit” maps were generatedfor bound ligands using phases from final models without ligand coordinatesafter application of small random shifts to the models and re-refining.The corresponding stereo images are shown in Figure S1 of the Supporting Information. Some amino acids interactingwith the ligands have been omitted for the sake of clarity, but allare shown in Figure S2 of the Supporting Information. Subsites −1 and −2 are labeled.

Mentions: To determine a structure with the intact donor substratebound,the predicted nucleophilic residue, Asp394, was mutated to Ala. Themaltosyl transferase activity of the D394A mutein was >4 ordersofmagnitude lower than that of the wild-type protein as expected. Acrystal of the D394A mutein was soaked in 5 mM α-maltose 1-phosphate,and a ligand-bound structure was successfully determined to 2.55 Ǻresolution (PDB entry 4CN1 in Figure 2A and Figure S1Cof the Supporting Information). The abilityto observe the intact substrate was consistent with a significantlyweaker ability of the mutein to catalyze its hydrolytic side reactionas expected. The substitution of Asp394 with Ala was confirmed inthe electron density map, but no other significant differences inthe protein structure were observed (Figures S1 and S2 of the Supporting Information). The interactions betweenthe protein and the sugar rings were very similar to those observedin the maltose-bound structures,10 andthe overall conformations of the sugar rings were likewise similar(Figure 3). This supports the assignment ofsubsites −2 and −1 and is consistent with the structuresof other GH13 enzymes.43 Therefore, thephosphate group of α-maltose 1-phosphate was located in whatwas predicted to be subsite +1.10 Thisgroup made hydrogen bonding interactions with the side chains of Asn352and Tyr357 from domain B and Asn395 and Glu423 from domain A, as previouslypredicted10 (Figure 2A and Figures S1C and S2B of the Supporting Information). The carboxyl group of Glu423 formed an ∼3.0 Å hydrogenbond to one of the oxygen atoms of the phosphate group and was only∼3.7 Å from the phosphate ester oxygen atom. The proximityof this conserved amino acid to the phosphate group is consistentwith it being the general acid/base catalytic residue that protonatesthe leaving group. The side chain of Glu423 also interacted with thebackbone NH group of Asn395, which could have a role in defining itspKa, a possibility that would requirefurther investigation. Interestingly, the phosphate does not sit inthe “tucked under” conformation seen for GT35 phosphorylasessuch as glycogen phosphorylase as well as many nucleotide sugar-dependentGTs.44,45 This difference likely reflects the quitedifferent mechanisms followed: double-SN2 displacementversus internal nucleophilic substitution SNi.46 While it is tempting to speculatefurther about the existence and role of each hydrogen bonding interactionobserved in this ligand-bound structure with a mutated enzyme, thehydrogen bonding network may be a little different in the true Michaeliscomplex.


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)

Structures of GlgE with (A) α-maltose 1-phosphate bound tothe D394A mutein (PDB entry 4CN1) and (B) the covalent intermediate formed betweenthe E423A mutein and 2-deoxy-2-fluoro-α-maltosyl fluoride (PDBentry 4CN4).Difference electron density “omit” maps were generatedfor bound ligands using phases from final models without ligand coordinatesafter application of small random shifts to the models and re-refining.The corresponding stereo images are shown in Figure S1 of the Supporting Information. Some amino acids interactingwith the ligands have been omitted for the sake of clarity, but allare shown in Figure S2 of the Supporting Information. Subsites −1 and −2 are labeled.
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fig2: Structures of GlgE with (A) α-maltose 1-phosphate bound tothe D394A mutein (PDB entry 4CN1) and (B) the covalent intermediate formed betweenthe E423A mutein and 2-deoxy-2-fluoro-α-maltosyl fluoride (PDBentry 4CN4).Difference electron density “omit” maps were generatedfor bound ligands using phases from final models without ligand coordinatesafter application of small random shifts to the models and re-refining.The corresponding stereo images are shown in Figure S1 of the Supporting Information. Some amino acids interactingwith the ligands have been omitted for the sake of clarity, but allare shown in Figure S2 of the Supporting Information. Subsites −1 and −2 are labeled.
Mentions: To determine a structure with the intact donor substratebound,the predicted nucleophilic residue, Asp394, was mutated to Ala. Themaltosyl transferase activity of the D394A mutein was >4 ordersofmagnitude lower than that of the wild-type protein as expected. Acrystal of the D394A mutein was soaked in 5 mM α-maltose 1-phosphate,and a ligand-bound structure was successfully determined to 2.55 Ǻresolution (PDB entry 4CN1 in Figure 2A and Figure S1Cof the Supporting Information). The abilityto observe the intact substrate was consistent with a significantlyweaker ability of the mutein to catalyze its hydrolytic side reactionas expected. The substitution of Asp394 with Ala was confirmed inthe electron density map, but no other significant differences inthe protein structure were observed (Figures S1 and S2 of the Supporting Information). The interactions betweenthe protein and the sugar rings were very similar to those observedin the maltose-bound structures,10 andthe overall conformations of the sugar rings were likewise similar(Figure 3). This supports the assignment ofsubsites −2 and −1 and is consistent with the structuresof other GH13 enzymes.43 Therefore, thephosphate group of α-maltose 1-phosphate was located in whatwas predicted to be subsite +1.10 Thisgroup made hydrogen bonding interactions with the side chains of Asn352and Tyr357 from domain B and Asn395 and Glu423 from domain A, as previouslypredicted10 (Figure 2A and Figures S1C and S2B of the Supporting Information). The carboxyl group of Glu423 formed an ∼3.0 Å hydrogenbond to one of the oxygen atoms of the phosphate group and was only∼3.7 Å from the phosphate ester oxygen atom. The proximityof this conserved amino acid to the phosphate group is consistentwith it being the general acid/base catalytic residue that protonatesthe leaving group. The side chain of Glu423 also interacted with thebackbone NH group of Asn395, which could have a role in defining itspKa, a possibility that would requirefurther investigation. Interestingly, the phosphate does not sit inthe “tucked under” conformation seen for GT35 phosphorylasessuch as glycogen phosphorylase as well as many nucleotide sugar-dependentGTs.44,45 This difference likely reflects the quitedifferent mechanisms followed: double-SN2 displacementversus internal nucleophilic substitution SNi.46 While it is tempting to speculatefurther about the existence and role of each hydrogen bonding interactionobserved in this ligand-bound structure with a mutated enzyme, thehydrogen bonding network may be a little different in the true Michaeliscomplex.

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