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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) Close view of domain B and S of opposing protomers of M. thermoresistibile GlgE with maltose bound. (B) View of the active site of M. thermoresistibile GlgE with maltose bound. Individual domains are represented in different colours. Dashed black lines represent hydrogen bonds. Subsites −1 and −2 are highlighted (C) Difference electron density map “omit map” of maltose. This map was generated using the phases from the final model.
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f2: (A) Close view of domain B and S of opposing protomers of M. thermoresistibile GlgE with maltose bound. (B) View of the active site of M. thermoresistibile GlgE with maltose bound. Individual domains are represented in different colours. Dashed black lines represent hydrogen bonds. Subsites −1 and −2 are highlighted (C) Difference electron density map “omit map” of maltose. This map was generated using the phases from the final model.

Mentions: M. thermoresistibile GlgE forms a dimer in all obtained structures and in solution, a feature reported previously for GlgE in other studied organisms4515. Each of the protomers of M. thermoresistibile GlgE contains 5 domains and 2 inserts (Fig. 1) that have been described extensively in the first reported GlgE structure15 and therefore we will focus on the particular characteristics of M. thermoresistibile GlgE. The N-terminal, domain N is a β-sandwich domain, responsible for the majority of dimerization contacts and interacting directly with the catalytic domain of the adjacent protomer. This domain contains a very long loop of 36 residues, connecting β-strands 4 and 5 of this domain, part of which (residues 71–86) has no observable density and therefore was not modelled. This loop is highly variable both in length and amino acid composition even among closely related mycobacterial species (Supplementary Fig. S2). Domains S and B also contribute to the dimer interface but to a lesser extent (Fig. 1 and Fig. 2A). Domain A and B together with inserts 1 and 2 form the catalytic unit. The C-terminal domain (domain C), a second β-sandwich domain, is not directly involved in catalytic activity but sits on top of the catalytic domain A, as reported before in S. coelicolor GlgE15. A C-terminal short α-helix in the M. thermoresistibile domain C, which contacts domain N, is found in all other mycobacterial GlgEs but is not found in that of S. coelicolor (Supplementary Fig. S2). Domain C together with domain A were recently shown to to be involved in the binding of α-glucan chains19. Although the identified binding patch in the surface of the protein is not 100% conserved there is a high degree of conservation among mycobacterial species (Supplementary Fig. S2).


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) Close view of domain B and S of opposing protomers of M. thermoresistibile GlgE with maltose bound. (B) View of the active site of M. thermoresistibile GlgE with maltose bound. Individual domains are represented in different colours. Dashed black lines represent hydrogen bonds. Subsites −1 and −2 are highlighted (C) Difference electron density map “omit map” of maltose. This map was generated using the phases from the final model.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f2: (A) Close view of domain B and S of opposing protomers of M. thermoresistibile GlgE with maltose bound. (B) View of the active site of M. thermoresistibile GlgE with maltose bound. Individual domains are represented in different colours. Dashed black lines represent hydrogen bonds. Subsites −1 and −2 are highlighted (C) Difference electron density map “omit map” of maltose. This map was generated using the phases from the final model.
Mentions: M. thermoresistibile GlgE forms a dimer in all obtained structures and in solution, a feature reported previously for GlgE in other studied organisms4515. Each of the protomers of M. thermoresistibile GlgE contains 5 domains and 2 inserts (Fig. 1) that have been described extensively in the first reported GlgE structure15 and therefore we will focus on the particular characteristics of M. thermoresistibile GlgE. The N-terminal, domain N is a β-sandwich domain, responsible for the majority of dimerization contacts and interacting directly with the catalytic domain of the adjacent protomer. This domain contains a very long loop of 36 residues, connecting β-strands 4 and 5 of this domain, part of which (residues 71–86) has no observable density and therefore was not modelled. This loop is highly variable both in length and amino acid composition even among closely related mycobacterial species (Supplementary Fig. S2). Domains S and B also contribute to the dimer interface but to a lesser extent (Fig. 1 and Fig. 2A). Domain A and B together with inserts 1 and 2 form the catalytic unit. The C-terminal domain (domain C), a second β-sandwich domain, is not directly involved in catalytic activity but sits on top of the catalytic domain A, as reported before in S. coelicolor GlgE15. A C-terminal short α-helix in the M. thermoresistibile domain C, which contacts domain N, is found in all other mycobacterial GlgEs but is not found in that of S. coelicolor (Supplementary Fig. S2). Domain C together with domain A were recently shown to to be involved in the binding of α-glucan chains19. Although the identified binding patch in the surface of the protein is not 100% conserved there is a high degree of conservation among mycobacterial species (Supplementary Fig. S2).

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