Structural analysis of phosphoribosyltransferase-mediated cell wall precursor synthesis in Mycobacterium tuberculosis

Shan Gao; Fangyu Wu; Sudagar S Gurcha; Sarah M Batt; Gurdyal S Besra; Zihe Rao; Lu Zhang

doi:10.1038/s41564-024-01643-8

Structural analysis of phosphoribosyltransferase-mediated cell wall precursor synthesis in Mycobacterium tuberculosis

Shan Gao, Fangyu Wu, Sudagar S Gurcha, Sarah M Batt, Gurdyal S Besra^*, Zihe Rao^*, Lu Zhang^*

^*Corresponding author for this work

Biosciences

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Abstract

In Mycobacterium tuberculosis, Rv3806c is a membrane-bound phosphoribosyltransferase (PRTase) involved in cell wall precursor production. It catalyses pentosyl phosphate transfer from phosphoribosyl pyrophosphate to decaprenyl phosphate, to generate 5-phospho-β-ribosyl-1-phosphoryldecaprenol. Despite Rv3806c being an attractive drug target, structural and molecular mechanistic insight into this PRTase is lacking. Here we report cryogenic electron microscopy structures for Rv3806c in the donor- and acceptor-bound states. In a lipidic environment, Rv3806c is trimeric, creating a UbiA-like fold. Each protomer forms two helical bundles, which, alongside the bound lipids, are required for PRTase activity in vitro. Mutational and functional analyses reveal that decaprenyl phosphate and phosphoribosyl pyrophosphate bind the intramembrane and extramembrane cavities of Rv3806c, respectively, in a distinct manner to that of UbiA superfamily enzymes. Our data suggest a model for Rv3806c-catalysed phosphoribose transfer through an inverting mechanism. These findings provide a structural basis for cell wall precursor biosynthesis that could have potential for anti-tuberculosis drug development.

Original language	English
Journal	Nature Microbiology
Early online date	15 Mar 2024
DOIs	https://doi.org/10.1038/s41564-024-01643-8
Publication status	E-pub ahead of print - 15 Mar 2024

Bibliographical note

Acknowledgments:
We thank W. Xu from ShanghaiTech University for discussion of the manuscript. We thank L. Wang and Q. Sun from the Bio-Electron Microscopy Facility and J. Chen, W. Zhu and X. Gao from Analytical Chemistry Platform of Shanghai Institute for Advanced Immunochemical Studies (SIAIS) of ShanghaiTech University for their support in EM data collection and mass spectrometry analysis respectively. We thank J. Liu at the Protein Preparation and Characterization Platform of Technology Center for Protein Research, Tsinghua University for providing facility support in MMS analysis. We thank J. Li from Large-scale Protein Preparation System at the National Facility for Protein Science in Shanghai (NFPS) for technical support. This work was supported by grants from National Natural Science Foundation of China (grant no. 32394010 to Z.R., and grant no. 32394011 to L.Z.), R&D Program of Guangzhou Laboratory (SRPG22-003) to Z.R. and L.Z., Shanghai Frontiers Science Center for Biomacromolecules and Precision Medicine, ShanghaiTech University, Medical Research Council UK (MR/S000542/1 and MR/R001154/1) to G.S.B.

Copyright:
© 2024. The Author(s).

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1 Finished
1 Active

The Mycobacterium tuberculosis Cell Envelope: unravelling complex cell wall assembly, degradation and re-cycling pathways
Besra, D. (Principal Investigator), Bhatt, A. (Co-Investigator), Futterer, K. (Co-Investigator), Alderwick, L. (Co-Investigator) & Zhang, J. (Co-Investigator)
Medical Research Council
1/03/19 → 28/02/25
Project: Research Councils
MICA: Addressing the burgeoning problem of tuberculosis: Exploiting phenotypic hits to identify new protein targets for drug discovery
Besra, D. (Principal Investigator), Cox, L. (Co-Investigator) & Futterer, K. (Co-Investigator)
Medical Research Council
1/04/18 → 31/03/22
Project: Research Councils

Cite this

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title = "Structural analysis of phosphoribosyltransferase-mediated cell wall precursor synthesis in Mycobacterium tuberculosis",

abstract = "In Mycobacterium tuberculosis, Rv3806c is a membrane-bound phosphoribosyltransferase (PRTase) involved in cell wall precursor production. It catalyses pentosyl phosphate transfer from phosphoribosyl pyrophosphate to decaprenyl phosphate, to generate 5-phospho-β-ribosyl-1-phosphoryldecaprenol. Despite Rv3806c being an attractive drug target, structural and molecular mechanistic insight into this PRTase is lacking. Here we report cryogenic electron microscopy structures for Rv3806c in the donor- and acceptor-bound states. In a lipidic environment, Rv3806c is trimeric, creating a UbiA-like fold. Each protomer forms two helical bundles, which, alongside the bound lipids, are required for PRTase activity in vitro. Mutational and functional analyses reveal that decaprenyl phosphate and phosphoribosyl pyrophosphate bind the intramembrane and extramembrane cavities of Rv3806c, respectively, in a distinct manner to that of UbiA superfamily enzymes. Our data suggest a model for Rv3806c-catalysed phosphoribose transfer through an inverting mechanism. These findings provide a structural basis for cell wall precursor biosynthesis that could have potential for anti-tuberculosis drug development.",

author = "Shan Gao and Fangyu Wu and Gurcha, {Sudagar S} and Batt, {Sarah M} and Besra, {Gurdyal S} and Zihe Rao and Lu Zhang",

note = "Acknowledgments: We thank W. Xu from ShanghaiTech University for discussion of the manuscript. We thank L. Wang and Q. Sun from the Bio-Electron Microscopy Facility and J. Chen, W. Zhu and X. Gao from Analytical Chemistry Platform of Shanghai Institute for Advanced Immunochemical Studies (SIAIS) of ShanghaiTech University for their support in EM data collection and mass spectrometry analysis respectively. We thank J. Liu at the Protein Preparation and Characterization Platform of Technology Center for Protein Research, Tsinghua University for providing facility support in MMS analysis. We thank J. Li from Large-scale Protein Preparation System at the National Facility for Protein Science in Shanghai (NFPS) for technical support. This work was supported by grants from National Natural Science Foundation of China (grant no. 32394010 to Z.R., and grant no. 32394011 to L.Z.), R&D Program of Guangzhou Laboratory (SRPG22-003) to Z.R. and L.Z., Shanghai Frontiers Science Center for Biomacromolecules and Precision Medicine, ShanghaiTech University, Medical Research Council UK (MR/S000542/1 and MR/R001154/1) to G.S.B. Copyright: {\textcopyright} 2024. The Author(s).",

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AU - Gao, Shan

AU - Wu, Fangyu

AU - Gurcha, Sudagar S

AU - Batt, Sarah M

AU - Besra, Gurdyal S

AU - Rao, Zihe

AU - Zhang, Lu

N1 - Acknowledgments: We thank W. Xu from ShanghaiTech University for discussion of the manuscript. We thank L. Wang and Q. Sun from the Bio-Electron Microscopy Facility and J. Chen, W. Zhu and X. Gao from Analytical Chemistry Platform of Shanghai Institute for Advanced Immunochemical Studies (SIAIS) of ShanghaiTech University for their support in EM data collection and mass spectrometry analysis respectively. We thank J. Liu at the Protein Preparation and Characterization Platform of Technology Center for Protein Research, Tsinghua University for providing facility support in MMS analysis. We thank J. Li from Large-scale Protein Preparation System at the National Facility for Protein Science in Shanghai (NFPS) for technical support. This work was supported by grants from National Natural Science Foundation of China (grant no. 32394010 to Z.R., and grant no. 32394011 to L.Z.), R&D Program of Guangzhou Laboratory (SRPG22-003) to Z.R. and L.Z., Shanghai Frontiers Science Center for Biomacromolecules and Precision Medicine, ShanghaiTech University, Medical Research Council UK (MR/S000542/1 and MR/R001154/1) to G.S.B. Copyright: © 2024. The Author(s).

PY - 2024/3/15

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N2 - In Mycobacterium tuberculosis, Rv3806c is a membrane-bound phosphoribosyltransferase (PRTase) involved in cell wall precursor production. It catalyses pentosyl phosphate transfer from phosphoribosyl pyrophosphate to decaprenyl phosphate, to generate 5-phospho-β-ribosyl-1-phosphoryldecaprenol. Despite Rv3806c being an attractive drug target, structural and molecular mechanistic insight into this PRTase is lacking. Here we report cryogenic electron microscopy structures for Rv3806c in the donor- and acceptor-bound states. In a lipidic environment, Rv3806c is trimeric, creating a UbiA-like fold. Each protomer forms two helical bundles, which, alongside the bound lipids, are required for PRTase activity in vitro. Mutational and functional analyses reveal that decaprenyl phosphate and phosphoribosyl pyrophosphate bind the intramembrane and extramembrane cavities of Rv3806c, respectively, in a distinct manner to that of UbiA superfamily enzymes. Our data suggest a model for Rv3806c-catalysed phosphoribose transfer through an inverting mechanism. These findings provide a structural basis for cell wall precursor biosynthesis that could have potential for anti-tuberculosis drug development.

AB - In Mycobacterium tuberculosis, Rv3806c is a membrane-bound phosphoribosyltransferase (PRTase) involved in cell wall precursor production. It catalyses pentosyl phosphate transfer from phosphoribosyl pyrophosphate to decaprenyl phosphate, to generate 5-phospho-β-ribosyl-1-phosphoryldecaprenol. Despite Rv3806c being an attractive drug target, structural and molecular mechanistic insight into this PRTase is lacking. Here we report cryogenic electron microscopy structures for Rv3806c in the donor- and acceptor-bound states. In a lipidic environment, Rv3806c is trimeric, creating a UbiA-like fold. Each protomer forms two helical bundles, which, alongside the bound lipids, are required for PRTase activity in vitro. Mutational and functional analyses reveal that decaprenyl phosphate and phosphoribosyl pyrophosphate bind the intramembrane and extramembrane cavities of Rv3806c, respectively, in a distinct manner to that of UbiA superfamily enzymes. Our data suggest a model for Rv3806c-catalysed phosphoribose transfer through an inverting mechanism. These findings provide a structural basis for cell wall precursor biosynthesis that could have potential for anti-tuberculosis drug development.

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DO - 10.1038/s41564-024-01643-8

M3 - Article

C2 - 38491273

SN - 2058-5276

JO - Nature Microbiology

JF - Nature Microbiology

ER -

Structural analysis of phosphoribosyltransferase-mediated cell wall precursor synthesis in Mycobacterium tuberculosis

Abstract

Bibliographical note

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Projects

The Mycobacterium tuberculosis Cell Envelope: unravelling complex cell wall assembly, degradation and re-cycling pathways

MICA: Addressing the burgeoning problem of tuberculosis: Exploiting phenotypic hits to identify new protein targets for drug discovery

Cite this