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Crystal structures of Mycobacterium tuberculosis GlgE and complexes with non-covalent inhibitors.

Lindenberger JJ, Veleti SK, Wilson BN, Sucheck SJ, Ronning DR - Sci Rep (2015)

Bottom Line: The maltohexaose structure reveals a dominant site for α-glucan binding.To obtain more detailed interactions between first generation, non-covalent inhibitors and GlgE, a variant Streptomyces coelicolor GlgEI (Sco GlgEI-V279S) was made to better emulate the Mtb GlgE M1P binding site.These structures detail important interactions that contribute to the inhibitory activity of these compounds, and provide information on future designs that may be exploited to improve upon these first generation GlgE inhibitors.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry and Biochemistry, The University of Toledo, 2801 W. Bancroft St. Ms602, Toledo, OH, United States.

ABSTRACT
GlgE is a bacterial maltosyltransferase that catalyzes the elongation of a cytosolic, branched α-glucan. In Mycobacterium tuberculosis (M. tb), inactivation of GlgE (Mtb GlgE) results in the rapid death of the organism due to a toxic accumulation of the maltosyl donor, maltose-1-phosphate (M1P), suggesting that GlgE is an intriguing target for inhibitor design. In this study, the crystal structures of the Mtb GlgE in a binary complex with maltose and a ternary complex with maltose and a maltosyl-acceptor molecule, maltohexaose, were solved to 3.3 Å and 4.0 Å, respectively. The maltohexaose structure reveals a dominant site for α-glucan binding. To obtain more detailed interactions between first generation, non-covalent inhibitors and GlgE, a variant Streptomyces coelicolor GlgEI (Sco GlgEI-V279S) was made to better emulate the Mtb GlgE M1P binding site. The structure of Sco GlgEI-V279S complexed with α-maltose-C-phosphonate (MCP), a non-hydrolyzable substrate analogue, was solved to 1.9 Å resolution, and the structure of Sco GlgEI-V279S complexed with 2,5-dideoxy-3-O-α-D-glucopyranosyl-2,5-imino-D-mannitol (DDGIM), an oxocarbenium mimic, was solved to 2.5 Å resolution. These structures detail important interactions that contribute to the inhibitory activity of these compounds, and provide information on future designs that may be exploited to improve upon these first generation GlgE inhibitors.

No MeSH data available.


Related in: MedlinePlus

Sco GlgEI-V279S in complex with MCP and DDGIM.(A) Fo-Fc omit map calculated while omitting MCP is contoured at 3σ with MCP bound in the enzyme active site. (B) Numerous hydrogen bonded interactions coordinate the glucosyl moiety in the −2 site, while the four residues in the phosphate-binding site coordinate the phosphonate moiety of the MCP. The D394/418 nucleophile is present in this structure and is positioned 3.3 Å from the C1′ of the MCP. E423/447 is well positioned to act as a general acid to protonate O1 of the phosphate of M1P at a distance of 3.8 Å. (C) Fo-Fc omit map calculated while omitting DDGIM is contoured at 3σ showing the DDGIM bound in the enzyme active site. (D) The glucose moiety of the DDGIM in the −2 site is coordinated in the same manner as was observed in the previous structures. Interactions of the imino mannitol in the −1 site are numerous. The O01 hydroxyl is hydrogen bonded with Q324/448, the general acid E423/447 is forming a hydrogen bond via the O01 hydroxyl of the imino mannitol, as well as an additional hydrogen bond between D480/504 and O09 hydroxyl. D394/418 is forming an ionic interaction with the secondary ammonium of the DDGIM. Inset between each image is the line structure of each inhibitor.
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f4: Sco GlgEI-V279S in complex with MCP and DDGIM.(A) Fo-Fc omit map calculated while omitting MCP is contoured at 3σ with MCP bound in the enzyme active site. (B) Numerous hydrogen bonded interactions coordinate the glucosyl moiety in the −2 site, while the four residues in the phosphate-binding site coordinate the phosphonate moiety of the MCP. The D394/418 nucleophile is present in this structure and is positioned 3.3 Å from the C1′ of the MCP. E423/447 is well positioned to act as a general acid to protonate O1 of the phosphate of M1P at a distance of 3.8 Å. (C) Fo-Fc omit map calculated while omitting DDGIM is contoured at 3σ showing the DDGIM bound in the enzyme active site. (D) The glucose moiety of the DDGIM in the −2 site is coordinated in the same manner as was observed in the previous structures. Interactions of the imino mannitol in the −1 site are numerous. The O01 hydroxyl is hydrogen bonded with Q324/448, the general acid E423/447 is forming a hydrogen bond via the O01 hydroxyl of the imino mannitol, as well as an additional hydrogen bond between D480/504 and O09 hydroxyl. D394/418 is forming an ionic interaction with the secondary ammonium of the DDGIM. Inset between each image is the line structure of each inhibitor.

Mentions: Since GlgE uses M1P as a maltosyl donor to extend the glucan, it was hypothesized that a non-hydrolyzable substrate analog could potentially function as a competitive inhibitor of GlgE. This compound, α-maltose-C-phosphonate (MCP), was synthesized and tested for GlgE inhibitory activity (Fig. 1C)16. MCP inhibited the Mtb GlgE with an IC50 of 237 ± 24 μM, which is roughly equivalent to the KM of the natural M1P substrate. This compound was co-crystallized with the Sco GlgEI-V279S to characterize the molecular basis of this inhibition. The resulting 1.9 Å resolution structure (Table 1) exhibits difference density within the M1P binding site that clearly illustrates the binding mode of MCP (Fig. 4A).


Crystal structures of Mycobacterium tuberculosis GlgE and complexes with non-covalent inhibitors.

Lindenberger JJ, Veleti SK, Wilson BN, Sucheck SJ, Ronning DR - Sci Rep (2015)

Sco GlgEI-V279S in complex with MCP and DDGIM.(A) Fo-Fc omit map calculated while omitting MCP is contoured at 3σ with MCP bound in the enzyme active site. (B) Numerous hydrogen bonded interactions coordinate the glucosyl moiety in the −2 site, while the four residues in the phosphate-binding site coordinate the phosphonate moiety of the MCP. The D394/418 nucleophile is present in this structure and is positioned 3.3 Å from the C1′ of the MCP. E423/447 is well positioned to act as a general acid to protonate O1 of the phosphate of M1P at a distance of 3.8 Å. (C) Fo-Fc omit map calculated while omitting DDGIM is contoured at 3σ showing the DDGIM bound in the enzyme active site. (D) The glucose moiety of the DDGIM in the −2 site is coordinated in the same manner as was observed in the previous structures. Interactions of the imino mannitol in the −1 site are numerous. The O01 hydroxyl is hydrogen bonded with Q324/448, the general acid E423/447 is forming a hydrogen bond via the O01 hydroxyl of the imino mannitol, as well as an additional hydrogen bond between D480/504 and O09 hydroxyl. D394/418 is forming an ionic interaction with the secondary ammonium of the DDGIM. Inset between each image is the line structure of each inhibitor.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4526890&req=5

f4: Sco GlgEI-V279S in complex with MCP and DDGIM.(A) Fo-Fc omit map calculated while omitting MCP is contoured at 3σ with MCP bound in the enzyme active site. (B) Numerous hydrogen bonded interactions coordinate the glucosyl moiety in the −2 site, while the four residues in the phosphate-binding site coordinate the phosphonate moiety of the MCP. The D394/418 nucleophile is present in this structure and is positioned 3.3 Å from the C1′ of the MCP. E423/447 is well positioned to act as a general acid to protonate O1 of the phosphate of M1P at a distance of 3.8 Å. (C) Fo-Fc omit map calculated while omitting DDGIM is contoured at 3σ showing the DDGIM bound in the enzyme active site. (D) The glucose moiety of the DDGIM in the −2 site is coordinated in the same manner as was observed in the previous structures. Interactions of the imino mannitol in the −1 site are numerous. The O01 hydroxyl is hydrogen bonded with Q324/448, the general acid E423/447 is forming a hydrogen bond via the O01 hydroxyl of the imino mannitol, as well as an additional hydrogen bond between D480/504 and O09 hydroxyl. D394/418 is forming an ionic interaction with the secondary ammonium of the DDGIM. Inset between each image is the line structure of each inhibitor.
Mentions: Since GlgE uses M1P as a maltosyl donor to extend the glucan, it was hypothesized that a non-hydrolyzable substrate analog could potentially function as a competitive inhibitor of GlgE. This compound, α-maltose-C-phosphonate (MCP), was synthesized and tested for GlgE inhibitory activity (Fig. 1C)16. MCP inhibited the Mtb GlgE with an IC50 of 237 ± 24 μM, which is roughly equivalent to the KM of the natural M1P substrate. This compound was co-crystallized with the Sco GlgEI-V279S to characterize the molecular basis of this inhibition. The resulting 1.9 Å resolution structure (Table 1) exhibits difference density within the M1P binding site that clearly illustrates the binding mode of MCP (Fig. 4A).

Bottom Line: The maltohexaose structure reveals a dominant site for α-glucan binding.To obtain more detailed interactions between first generation, non-covalent inhibitors and GlgE, a variant Streptomyces coelicolor GlgEI (Sco GlgEI-V279S) was made to better emulate the Mtb GlgE M1P binding site.These structures detail important interactions that contribute to the inhibitory activity of these compounds, and provide information on future designs that may be exploited to improve upon these first generation GlgE inhibitors.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry and Biochemistry, The University of Toledo, 2801 W. Bancroft St. Ms602, Toledo, OH, United States.

ABSTRACT
GlgE is a bacterial maltosyltransferase that catalyzes the elongation of a cytosolic, branched α-glucan. In Mycobacterium tuberculosis (M. tb), inactivation of GlgE (Mtb GlgE) results in the rapid death of the organism due to a toxic accumulation of the maltosyl donor, maltose-1-phosphate (M1P), suggesting that GlgE is an intriguing target for inhibitor design. In this study, the crystal structures of the Mtb GlgE in a binary complex with maltose and a ternary complex with maltose and a maltosyl-acceptor molecule, maltohexaose, were solved to 3.3 Å and 4.0 Å, respectively. The maltohexaose structure reveals a dominant site for α-glucan binding. To obtain more detailed interactions between first generation, non-covalent inhibitors and GlgE, a variant Streptomyces coelicolor GlgEI (Sco GlgEI-V279S) was made to better emulate the Mtb GlgE M1P binding site. The structure of Sco GlgEI-V279S complexed with α-maltose-C-phosphonate (MCP), a non-hydrolyzable substrate analogue, was solved to 1.9 Å resolution, and the structure of Sco GlgEI-V279S complexed with 2,5-dideoxy-3-O-α-D-glucopyranosyl-2,5-imino-D-mannitol (DDGIM), an oxocarbenium mimic, was solved to 2.5 Å resolution. These structures detail important interactions that contribute to the inhibitory activity of these compounds, and provide information on future designs that may be exploited to improve upon these first generation GlgE inhibitors.

No MeSH data available.


Related in: MedlinePlus