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Structure of Mycobacterium thermoresistibile GlgE defines novel conformational states that contribute to the catalytic mechanism.

Mendes V, Blaszczyk M, Maranha A, Empadinhas N, Blundell TL - Sci Rep (2015)

Bottom Line: Inhibition of GlgE, which transfers maltose from a maltose-1-phosphate donor to α-glucan/maltooligosaccharide chain acceptor, leads to a toxic accumulation of maltose-1-phosphate that culminates in cellular death.However, in M. thermoresistibile GlgE we observe several conformational states of the S domain and provide evidence that its high flexibility is important for enzyme activity.The structures here reported shed further light on the interactions between the N-terminal domains and the catalytic domains of opposing chains and how they contribute to the catalytic reaction.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, University of Cambridge, Cambridge CB2 1GA, UK.

ABSTRACT
GlgE, an enzyme of the pathway that converts trehalose to α-glucans, is essential for Mycobacterium tuberculosis. Inhibition of GlgE, which transfers maltose from a maltose-1-phosphate donor to α-glucan/maltooligosaccharide chain acceptor, leads to a toxic accumulation of maltose-1-phosphate that culminates in cellular death. Here we describe the first high-resolution mycobacterial GlgE structure from Mycobacterium thermoresistibile at 1.96 Å. We show that the structure resembles that of M. tuberculosis and Streptomyces coelicolor GlgEs, reported before, with each protomer in the homodimer comprising five domains. However, in M. thermoresistibile GlgE we observe several conformational states of the S domain and provide evidence that its high flexibility is important for enzyme activity. The structures here reported shed further light on the interactions between the N-terminal domains and the catalytic domains of opposing chains and how they contribute to the catalytic reaction. Importantly this work identifies a useful surrogate system to aid the development of GlgE inhibitors against opportunistic and pathogenic mycobacteria.

No MeSH data available.


Related in: MedlinePlus

(A) View of M. tuberculosis GlgE phosphorylation sites mapped in M. thermoresistibile GlgE structure (5GCM). T46 and T186 are conserved in M. tuberculosis and M. thermoresistibile. S143 and S365 are mutated to threonine in M. tuberculosis. A188 and M80 are mutated to serine in M. tuberculosis. The side chain of S143 and the loop where M80 is located were not modeled since electron density is poor in those regions. The putative phosphorylation site T98 and T445 are also highlighted. (B) Superposition of structures of maltose co-crystallization (5GCM), maltose-1P co-crystallization (5ICM) and apo form (5CJ5). The represented protomer of maltose-1P structure has maltose bound. Regions that lose helical conformation in the maltose-1P co-crystallization structure are highlighted in red.
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f3: (A) View of M. tuberculosis GlgE phosphorylation sites mapped in M. thermoresistibile GlgE structure (5GCM). T46 and T186 are conserved in M. tuberculosis and M. thermoresistibile. S143 and S365 are mutated to threonine in M. tuberculosis. A188 and M80 are mutated to serine in M. tuberculosis. The side chain of S143 and the loop where M80 is located were not modeled since electron density is poor in those regions. The putative phosphorylation site T98 and T445 are also highlighted. (B) Superposition of structures of maltose co-crystallization (5GCM), maltose-1P co-crystallization (5ICM) and apo form (5CJ5). The represented protomer of maltose-1P structure has maltose bound. Regions that lose helical conformation in the maltose-1P co-crystallization structure are highlighted in red.

Mentions: In the maltose co-crystallization structure (5CGM) several ethylene glycol molecules and polyethylene glycol chains of different sizes are visible due to PEG300 being present in the crystallization condition at high concentration (30%). Two phosphate groups can also be seen bound to each of the chains sitting in a groove between domain N and the terminal α-helix of C. Two other phosphate groups sit in a positively charged patch formed by domains A and N of the two opposing protomers, near the active site. Interestingly all the phosphate groups are close to threonines, T98 for the first duo and T445 for the second (Fig. 3).


Structure of Mycobacterium thermoresistibile GlgE defines novel conformational states that contribute to the catalytic mechanism.

Mendes V, Blaszczyk M, Maranha A, Empadinhas N, Blundell TL - Sci Rep (2015)

(A) View of M. tuberculosis GlgE phosphorylation sites mapped in M. thermoresistibile GlgE structure (5GCM). T46 and T186 are conserved in M. tuberculosis and M. thermoresistibile. S143 and S365 are mutated to threonine in M. tuberculosis. A188 and M80 are mutated to serine in M. tuberculosis. The side chain of S143 and the loop where M80 is located were not modeled since electron density is poor in those regions. The putative phosphorylation site T98 and T445 are also highlighted. (B) Superposition of structures of maltose co-crystallization (5GCM), maltose-1P co-crystallization (5ICM) and apo form (5CJ5). The represented protomer of maltose-1P structure has maltose bound. Regions that lose helical conformation in the maltose-1P co-crystallization structure are highlighted in red.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4663749&req=5

f3: (A) View of M. tuberculosis GlgE phosphorylation sites mapped in M. thermoresistibile GlgE structure (5GCM). T46 and T186 are conserved in M. tuberculosis and M. thermoresistibile. S143 and S365 are mutated to threonine in M. tuberculosis. A188 and M80 are mutated to serine in M. tuberculosis. The side chain of S143 and the loop where M80 is located were not modeled since electron density is poor in those regions. The putative phosphorylation site T98 and T445 are also highlighted. (B) Superposition of structures of maltose co-crystallization (5GCM), maltose-1P co-crystallization (5ICM) and apo form (5CJ5). The represented protomer of maltose-1P structure has maltose bound. Regions that lose helical conformation in the maltose-1P co-crystallization structure are highlighted in red.
Mentions: In the maltose co-crystallization structure (5CGM) several ethylene glycol molecules and polyethylene glycol chains of different sizes are visible due to PEG300 being present in the crystallization condition at high concentration (30%). Two phosphate groups can also be seen bound to each of the chains sitting in a groove between domain N and the terminal α-helix of C. Two other phosphate groups sit in a positively charged patch formed by domains A and N of the two opposing protomers, near the active site. Interestingly all the phosphate groups are close to threonines, T98 for the first duo and T445 for the second (Fig. 3).

Bottom Line: Inhibition of GlgE, which transfers maltose from a maltose-1-phosphate donor to α-glucan/maltooligosaccharide chain acceptor, leads to a toxic accumulation of maltose-1-phosphate that culminates in cellular death.However, in M. thermoresistibile GlgE we observe several conformational states of the S domain and provide evidence that its high flexibility is important for enzyme activity.The structures here reported shed further light on the interactions between the N-terminal domains and the catalytic domains of opposing chains and how they contribute to the catalytic reaction.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, University of Cambridge, Cambridge CB2 1GA, UK.

ABSTRACT
GlgE, an enzyme of the pathway that converts trehalose to α-glucans, is essential for Mycobacterium tuberculosis. Inhibition of GlgE, which transfers maltose from a maltose-1-phosphate donor to α-glucan/maltooligosaccharide chain acceptor, leads to a toxic accumulation of maltose-1-phosphate that culminates in cellular death. Here we describe the first high-resolution mycobacterial GlgE structure from Mycobacterium thermoresistibile at 1.96 Å. We show that the structure resembles that of M. tuberculosis and Streptomyces coelicolor GlgEs, reported before, with each protomer in the homodimer comprising five domains. However, in M. thermoresistibile GlgE we observe several conformational states of the S domain and provide evidence that its high flexibility is important for enzyme activity. The structures here reported shed further light on the interactions between the N-terminal domains and the catalytic domains of opposing chains and how they contribute to the catalytic reaction. Importantly this work identifies a useful surrogate system to aid the development of GlgE inhibitors against opportunistic and pathogenic mycobacteria.

No MeSH data available.


Related in: MedlinePlus