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Structures of DPAGT1 explain glycosylation disease mechanisms and advance TB antibiotic design

View ORCID ProfileYin Yao Dong, Hua Wang, Ashley C.W. Pike, Stephen A. Cochrane, Sadra Hamedzadeh, Filip J. Wyszyński, Simon R. Bushell, Sylvain F. Royer, David A. Widdick, Andaleeb Sajid, Helena I. Boshoff, Ricardo Lucas, Wei-Min Liu, Seung Seo Lee, Takuya Machida, Shahid Mehmood, Katsiaryna Belaya, Wei-Wei Liu, Amy Chu, Leela Shrestha, Shubhashish M.M. Mukhopadhyay, Nicola A. Burgess-Brown, Mervyn J. Bibb, Clifton E. Barry 3rd, Carol V. Robinson, David Beeson, Benjamin G. Davis, Elisabeth P. Carpenter
doi: https://doi.org/10.1101/291278
Yin Yao Dong
1Structural Genomics Consortium, University of Oxford, Oxford, OX3 7DQ, UK.
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Hua Wang
2Chemistry Research Laboratory, University of Oxford, 12 Mansfield Road, Oxford OX1 3TA, UK.
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Ashley C.W. Pike
1Structural Genomics Consortium, University of Oxford, Oxford, OX3 7DQ, UK.
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Stephen A. Cochrane
2Chemistry Research Laboratory, University of Oxford, 12 Mansfield Road, Oxford OX1 3TA, UK.
3School of Chemistry and Chemical Engineering, Queen’s University Belfast, UK.
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Sadra Hamedzadeh
2Chemistry Research Laboratory, University of Oxford, 12 Mansfield Road, Oxford OX1 3TA, UK.
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Filip J. Wyszyński
2Chemistry Research Laboratory, University of Oxford, 12 Mansfield Road, Oxford OX1 3TA, UK.
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Simon R. Bushell
1Structural Genomics Consortium, University of Oxford, Oxford, OX3 7DQ, UK.
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Sylvain F. Royer
2Chemistry Research Laboratory, University of Oxford, 12 Mansfield Road, Oxford OX1 3TA, UK.
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David A. Widdick
4Department of Molecular Microbiology, John Innes Centre, Norwich Research Park, Norwich NR4 7UH, UK.
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Andaleeb Sajid
5Tuberculosis Research Section, Laboratory of Clinical Immunology and Microbiology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland 20892, USA.
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Helena I. Boshoff
5Tuberculosis Research Section, Laboratory of Clinical Immunology and Microbiology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland 20892, USA.
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Ricardo Lucas
2Chemistry Research Laboratory, University of Oxford, 12 Mansfield Road, Oxford OX1 3TA, UK.
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Wei-Min Liu
2Chemistry Research Laboratory, University of Oxford, 12 Mansfield Road, Oxford OX1 3TA, UK.
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Seung Seo Lee
2Chemistry Research Laboratory, University of Oxford, 12 Mansfield Road, Oxford OX1 3TA, UK.
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Takuya Machida
2Chemistry Research Laboratory, University of Oxford, 12 Mansfield Road, Oxford OX1 3TA, UK.
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Shahid Mehmood
6Department of Chemistry, South Parks Road, Oxford OX1 3QZ UK.
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Katsiaryna Belaya
7Neurosciences Group, Nuffield Department of Clinical Neuroscience, Weatherall Institute of Molecular Medicine, University of Oxford, Oxford, OX3 9DS, UK.
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Wei-Wei Liu
7Neurosciences Group, Nuffield Department of Clinical Neuroscience, Weatherall Institute of Molecular Medicine, University of Oxford, Oxford, OX3 9DS, UK.
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Amy Chu
1Structural Genomics Consortium, University of Oxford, Oxford, OX3 7DQ, UK.
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Leela Shrestha
1Structural Genomics Consortium, University of Oxford, Oxford, OX3 7DQ, UK.
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Shubhashish M.M. Mukhopadhyay
1Structural Genomics Consortium, University of Oxford, Oxford, OX3 7DQ, UK.
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Nicola A. Burgess-Brown
1Structural Genomics Consortium, University of Oxford, Oxford, OX3 7DQ, UK.
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Mervyn J. Bibb
4Department of Molecular Microbiology, John Innes Centre, Norwich Research Park, Norwich NR4 7UH, UK.
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Clifton E. Barry 3rd
5Tuberculosis Research Section, Laboratory of Clinical Immunology and Microbiology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland 20892, USA.
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Carol V. Robinson
6Department of Chemistry, South Parks Road, Oxford OX1 3QZ UK.
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David Beeson
7Neurosciences Group, Nuffield Department of Clinical Neuroscience, Weatherall Institute of Molecular Medicine, University of Oxford, Oxford, OX3 9DS, UK.
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Benjamin G. Davis
2Chemistry Research Laboratory, University of Oxford, 12 Mansfield Road, Oxford OX1 3TA, UK.
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  • For correspondence: Ben.Davis@chem.ox.ac.uk liz.carpenter@sgc.ox.ac.uk
Elisabeth P. Carpenter
1Structural Genomics Consortium, University of Oxford, Oxford, OX3 7DQ, UK.
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  • For correspondence: Ben.Davis@chem.ox.ac.uk liz.carpenter@sgc.ox.ac.uk
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Summary

Protein glycosylation is a widespread post-translational modification. The first committed step to the lipid-linked glycan used for this process is catalysed by dolichyl-phosphate N-acetylglucosamine-phosphotransferase DPAGT1 (GPT/E.C. 2.7.8.15). Missense DPAGT1 variants cause congenital myasthenic syndrome and congenital disorders of glycosylation. In addition, naturally-occurring bactericidal nucleoside analogues such as tunicamycin are toxic to eukaryotes due to DPAGT1 inhibition, preventing their clinical use as antibiotics. However, little is known about the mechanism or the effects of disease-associated mutations in this essential enzyme. Our structures of DPAGT1 with the substrate UDP-GlcNAc and tunicamycin reveal substrate binding modes, suggest a mechanism of catalysis, provide an understanding of how mutations modulate activity (and thus cause disease) and allow design of non-toxic ‘lipid-altered’ tunicamycins. The structure-tuned activity of these analogues against several bacterial targets allowed design of potent antibiotics for Mycobacterium tuberculosis, enabling treatment in vitro, in cellulo and in vivo thereby providing a promising new class of antimicrobial drug.

Highlights

  • Structures of DPAGT1 with UDP-GlcNAc and tunicamycin reveal mechanisms of catalysis

  • DPAGT1 mutants in patients with glycosylation disorders modulate DPAGT1 activity

  • Structures, kinetics and biosynthesis reveal role of lipid in tunicamycin

  • Lipid-altered, tunicamycin analogues give non-toxic antibiotics against TB

Copyright 
The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-NC-ND 4.0 International license.
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Posted April 02, 2018.
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Structures of DPAGT1 explain glycosylation disease mechanisms and advance TB antibiotic design
Yin Yao Dong, Hua Wang, Ashley C.W. Pike, Stephen A. Cochrane, Sadra Hamedzadeh, Filip J. Wyszyński, Simon R. Bushell, Sylvain F. Royer, David A. Widdick, Andaleeb Sajid, Helena I. Boshoff, Ricardo Lucas, Wei-Min Liu, Seung Seo Lee, Takuya Machida, Shahid Mehmood, Katsiaryna Belaya, Wei-Wei Liu, Amy Chu, Leela Shrestha, Shubhashish M.M. Mukhopadhyay, Nicola A. Burgess-Brown, Mervyn J. Bibb, Clifton E. Barry 3rd, Carol V. Robinson, David Beeson, Benjamin G. Davis, Elisabeth P. Carpenter
bioRxiv 291278; doi: https://doi.org/10.1101/291278
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Structures of DPAGT1 explain glycosylation disease mechanisms and advance TB antibiotic design
Yin Yao Dong, Hua Wang, Ashley C.W. Pike, Stephen A. Cochrane, Sadra Hamedzadeh, Filip J. Wyszyński, Simon R. Bushell, Sylvain F. Royer, David A. Widdick, Andaleeb Sajid, Helena I. Boshoff, Ricardo Lucas, Wei-Min Liu, Seung Seo Lee, Takuya Machida, Shahid Mehmood, Katsiaryna Belaya, Wei-Wei Liu, Amy Chu, Leela Shrestha, Shubhashish M.M. Mukhopadhyay, Nicola A. Burgess-Brown, Mervyn J. Bibb, Clifton E. Barry 3rd, Carol V. Robinson, David Beeson, Benjamin G. Davis, Elisabeth P. Carpenter
bioRxiv 291278; doi: https://doi.org/10.1101/291278

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