Limits...
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

Comparison of the active sites of chain A and B of the maltose-1P co-crystallization condition.Maltose is only present in chain B. The antiparallel β-strand lid and loop 2 of domain B are only visible in chain B.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4663749&req=5

f4: Comparison of the active sites of chain A and B of the maltose-1P co-crystallization condition.Maltose is only present in chain B. The antiparallel β-strand lid and loop 2 of domain B are only visible in chain B.

Mentions: In an attempt to obtain a maltosyl-GlgE intermediate state, GlgE was co-crystalized with maltose-1P. We could not observe the maltosyl-GlgE intermediate but instead we found maltose bound to only one of the active sites of the dimer (Fig. 4). The fact that maltose was present instead of maltose-1P is most likely due to GlgE slowly degrading maltose-1P to maltose, since maltose-1P was free of maltose contamination and we confirmed its stability at different temperatures for long periods of time (Supplementary Fig. S4). Moreover others have also reported slow degradation of maltose-1P by S. coelicolor GlgE during the crystallization time span15. Significant conformational changes were however visible in the structure especially in the S domain of the protomer with bound maltose (Fig. 3B and Supplementary Fig. S5A). The S domain of this protomer exhibits conformational changes with several residues of α1 (V132–L136) and α2 (S146–150) losing their helical conformation (Fig. 3B). These residues are those closer to loop 2 of domain B of the “apo” protomer and the observed changes can indicate differences in the contacts between these two domains (Supplementary Fig. S5A). Moreover, all four helices comprising the S domain of the maltose-bound protomer were found to be shifted with a maximum distance of ~6.5 Å when compared to both the maltose co-crystallization and the apo structure. This brings domain S of the maltose containing protomer closer to loop 2 of domain B of the “apo” protomer (Fig. 3). These observations imply a mechanism where domain S of one protomer interacts with loop 2 of domain B of the opposing protomer to move the antiparallel β-strand lid that covers the active site into an open or closed conformation, allowing the substrate/product to enter/leave the active site. Unfortunately, the majority of domain B, including loop 2 and the antiparallel β-strand lid, is not seen clearly in the maltose-1P co-crystallization structure “apo-protomer” and thus it could not be modeled (Fig. 4 and Supplementary Fig. S5A).


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)

Comparison of the active sites of chain A and B of the maltose-1P co-crystallization condition.Maltose is only present in chain B. The antiparallel β-strand lid and loop 2 of domain B are only visible in chain B.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f4: Comparison of the active sites of chain A and B of the maltose-1P co-crystallization condition.Maltose is only present in chain B. The antiparallel β-strand lid and loop 2 of domain B are only visible in chain B.
Mentions: In an attempt to obtain a maltosyl-GlgE intermediate state, GlgE was co-crystalized with maltose-1P. We could not observe the maltosyl-GlgE intermediate but instead we found maltose bound to only one of the active sites of the dimer (Fig. 4). The fact that maltose was present instead of maltose-1P is most likely due to GlgE slowly degrading maltose-1P to maltose, since maltose-1P was free of maltose contamination and we confirmed its stability at different temperatures for long periods of time (Supplementary Fig. S4). Moreover others have also reported slow degradation of maltose-1P by S. coelicolor GlgE during the crystallization time span15. Significant conformational changes were however visible in the structure especially in the S domain of the protomer with bound maltose (Fig. 3B and Supplementary Fig. S5A). The S domain of this protomer exhibits conformational changes with several residues of α1 (V132–L136) and α2 (S146–150) losing their helical conformation (Fig. 3B). These residues are those closer to loop 2 of domain B of the “apo” protomer and the observed changes can indicate differences in the contacts between these two domains (Supplementary Fig. S5A). Moreover, all four helices comprising the S domain of the maltose-bound protomer were found to be shifted with a maximum distance of ~6.5 Å when compared to both the maltose co-crystallization and the apo structure. This brings domain S of the maltose containing protomer closer to loop 2 of domain B of the “apo” protomer (Fig. 3). These observations imply a mechanism where domain S of one protomer interacts with loop 2 of domain B of the opposing protomer to move the antiparallel β-strand lid that covers the active site into an open or closed conformation, allowing the substrate/product to enter/leave the active site. Unfortunately, the majority of domain B, including loop 2 and the antiparallel β-strand lid, is not seen clearly in the maltose-1P co-crystallization structure “apo-protomer” and thus it could not be modeled (Fig. 4 and Supplementary Fig. S5A).

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