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Biochemical and Structural Characterization of the Carbohydrate Transport Substrate-binding-protein SP0092
Published on: October 2, 2017
Deciphering the mannose transfer mechanism of mycobacterial PimE by molecular dynamics simulations
Gourab Bhattacharje1, Amit Ghosh2, Amit Kumar Das1
1Department of Biotechnology, Indian Institute of Technology Kharagpur, Kharagpur, West Midnapore, WB 721302, India.
Abstract:
Phosphatidyl-myo-inositol mannosides (PIMs), Lipomannan (LM), and Lipoarabinomannan (LAM) are essential components of the cell envelopes of mycobacteria. At the beginning of the biosynthesis of these compounds, phosphatidylinositol (PI) is mannosylated and acylated by various enzymes to produce Ac1/2PIM4, which is used to synthesize either Ac1/2PIM6 or LM/LAM. The protein PimE, a membrane-bound glycosyltransferase (GT-C), catalyzes the addition of a mannose group to Ac1PIM4 to produce Ac1PIM5, using polyprenolphosphate mannose (PPM) as the mannose donor. PimE-deleted Mycobacterium smegmatis (Msmeg) showed structural deformity and increased antibiotic and copper sensitivity. Despite knowing that the mutation D58A caused inactivity in Msmeg, how PimE catalyzes the transfer of mannose from PPM to Ac1/2PIM4 remains unknown. In this study, analyzing the AlphaFold structure of PimE revealed the presence of a tunnel through the D58 residue with two differently charged gates. Molecular docking suggested PPM binds to the hydrophobic tunnel gate, whereas Ac1PIM4 binds to the positively charged tunnel gate. Molecular dynamics (MD) simulations further demonstrated the critical roles of the residues N55, F87, L89, Y163, Q165, K197, L198, R251, F277, W324, H326, and I375 in binding PPM and Ac1PIM4. The mutation D58A caused a faster release of PPM from the catalytic tunnel, explaining the loss of PimE activity. Along with a hypothetical mechanism of mannose transfer by PimE, we also observe the presence of tunnels through a negatively charged aspartate or glutamate with two differently-charged gates among most GT-C enzymes. Common hydrophobic gates of GT-C enzymes probably harbor sugar donors, whereas, differently-charged tunnel gates accommodate various sugar-acceptors.
Insights
The glycosyltransferase PimE transfers mannose to mycobacterial cell envelope components. Its catalytic tunnel, featuring charged gates, binds substrates, with D58A mutations disrupting this process and explaining enzyme inactivity.
Area of Science:
- Biochemistry
- Structural Biology
- Microbiology
Background:
- Phosphatidyl-myo-inositol mannosides (PIMs), Lipomannan (LM), and Lipoarabinomannan (LAM) are vital for mycobacterial cell envelopes.
- PimE is a glycosyltransferase crucial for synthesizing PIMs, with its D58A mutation rendering it inactive.
Purpose of the Study:
- To elucidate the mechanism of mannose transfer by PimE.
- To understand the structural basis for PimE's activity and the role of the D58 residue.
Main Methods:
- AlphaFold structure prediction of PimE.
- Molecular docking to analyze substrate binding.
- Molecular dynamics (MD) simulations to study enzyme-substrate interactions.
- Analysis of D58A mutant activity.
Main Results:
- PimE possesses a tunnel with two distinct gates, facilitating substrate binding.
- Molecular docking and MD simulations identified key residues involved in binding polyprenolphosphate mannose (PPM) and Ac1PIM4.
- The D58A mutation accelerates PPM release, explaining the loss of catalytic activity.
- A conserved tunnel structure with charged gates was observed in other GT-C enzymes.
Conclusions:
- PimE utilizes a catalytic tunnel with charged gates to bind mannose donors (PPM) and acceptors (Ac1PIM4).
- The D58 residue is critical for maintaining substrate binding within the catalytic tunnel.
- The findings provide a hypothetical mechanism for mannose transfer by PimE and suggest a conserved mechanism across GT-C enzymes.
