Heme-binding enables allosteric modulation in an ancient TIM-barrel glycosidase

Gloria Gamiz-Arco; Luis I. Gutierrez-Rus; Valeria A. Risso; Beatriz Ibarra-Molero; Yosuke Hoshino; Dušan Petrović; Jose Justicia; Juan Manuel Cuerva; Adrian Romero-Rivera; Burckhard Seelig; Jose A. Gavira; Shina C.L. Kamerlin; Eric A. Gaucher; Jose M. Sanchez-Ruiz

doi:10.1038/s41467-020-20630-1

Heme-binding enables allosteric modulation in an ancient TIM-barrel glycosidase

Gloria Gamiz-Arco, Luis I. Gutierrez-Rus, Valeria A. Risso, Beatriz Ibarra-Molero, Yosuke Hoshino, Dušan Petrović, Jose Justicia, Juan Manuel Cuerva, Adrian Romero-Rivera, Burckhard Seelig, Jose A. Gavira, Shina C.L. Kamerlin, Eric A. Gaucher, Jose M. Sanchez-Ruiz

Synthetic Biology and Biotechnology Division

Research output: Contribution to journal › Article › peer-review

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Abstract

Glycosidases are phylogenetically widely distributed enzymes that are crucial for the cleavage of glycosidic bonds. Here, we present the exceptional properties of a putative ancestor of bacterial and eukaryotic family-1 glycosidases. The ancestral protein shares the TIM-barrel fold with its modern descendants but displays large regions with greatly enhanced conformational flexibility. Yet, the barrel core remains comparatively rigid and the ancestral glycosidase activity is stable, with an optimum temperature within the experimental range for thermophilic family-1 glycosidases. None of the ∼5500 reported crystallographic structures of ∼1400 modern glycosidases show a bound porphyrin. Remarkably, the ancestral glycosidase binds heme tightly and stoichiometrically at a well-defined buried site. Heme binding rigidifies this TIM-barrel and allosterically enhances catalysis. Our work demonstrates the capability of ancestral protein reconstructions to reveal valuable but unexpected biomolecular features when sampling distant sequence space. The potential of the ancestral glycosidase as a scaffold for custom catalysis and biosensor engineering is discussed.

Original language	English (US)
Article number	380
Journal	Nature communications
Volume	12
Issue number	1
DOIs	https://doi.org/10.1038/s41467-020-20630-1
State	Published - Dec 1 2021

Bibliographical note

Funding Information:
This work was supported by Human Frontier Science Program Grant RGP0041 (J.M.S.-R., E.A.G., B.S., and S.C.L.K.), NIH grant R01AR069137 (E.A.G.), Department of Defense grant MURI W911NF-16-1-0372 (E.A.G.), the Swedish Research Council (2019-03499) (S.C.L.K.), the Knut and Alice Wallenberg Foundation (2018.0140 and 2019.0431) (S.C.L.K.), Spanish Ministry of Economy and Competitiveness/FEDER Funds Grants BIO2015-66426-R (J.M.S.-R.) RTI2018-097142-B-100 (J.M.S.-R.) and BIO2016-74875-P (J.A.G.). The simulations were enabled by resources provided by the Swedish National Infrastructure for Computing (SNIC) at UPPMAX partially funded by the Swedish Research Council through grant agreement no. 2016-07213. We acknowledge the Spanish Synchrotron Radiation Facility (ALBA, Barcelona) for the provision of synchrotron radiation facilities and the staff at XALOC beamline for their invaluable support. We are also grateful to Victoria Longobardo Polanco (Proteomic Unit, Institute of Parasitology and Biomedicine “López-Neyra”) for help with mass spectrometry experiments and data analyses and to Juan Román Luque Ortega (Molecular Interactions Facility, Centro de Investigaciones Biológicas Margarita Salas) for help with ultracentrifugation experiments and data analyses.

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Gamiz-Arco, G., Gutierrez-Rus, L. I., Risso, V. A., Ibarra-Molero, B., Hoshino, Y., Petrović, D., Justicia, J., Cuerva, J. M., Romero-Rivera, A., Seelig, B., Gavira, J. A., Kamerlin, S. C. L., Gaucher, E. A., & Sanchez-Ruiz, J. M. (2021). Heme-binding enables allosteric modulation in an ancient TIM-barrel glycosidase. Nature communications, 12(1), Article 380. https://doi.org/10.1038/s41467-020-20630-1

Gamiz-Arco, G, Gutierrez-Rus, LI, Risso, VA, Ibarra-Molero, B, Hoshino, Y, Petrović, D, Justicia, J, Cuerva, JM, Romero-Rivera, A, Seelig, B, Gavira, JA, Kamerlin, SCL, Gaucher, EA & Sanchez-Ruiz, JM 2021, 'Heme-binding enables allosteric modulation in an ancient TIM-barrel glycosidase', Nature communications, vol. 12, no. 1, 380. https://doi.org/10.1038/s41467-020-20630-1

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title = "Heme-binding enables allosteric modulation in an ancient TIM-barrel glycosidase",

abstract = "Glycosidases are phylogenetically widely distributed enzymes that are crucial for the cleavage of glycosidic bonds. Here, we present the exceptional properties of a putative ancestor of bacterial and eukaryotic family-1 glycosidases. The ancestral protein shares the TIM-barrel fold with its modern descendants but displays large regions with greatly enhanced conformational flexibility. Yet, the barrel core remains comparatively rigid and the ancestral glycosidase activity is stable, with an optimum temperature within the experimental range for thermophilic family-1 glycosidases. None of the ∼5500 reported crystallographic structures of ∼1400 modern glycosidases show a bound porphyrin. Remarkably, the ancestral glycosidase binds heme tightly and stoichiometrically at a well-defined buried site. Heme binding rigidifies this TIM-barrel and allosterically enhances catalysis. Our work demonstrates the capability of ancestral protein reconstructions to reveal valuable but unexpected biomolecular features when sampling distant sequence space. The potential of the ancestral glycosidase as a scaffold for custom catalysis and biosensor engineering is discussed.",

author = "Gloria Gamiz-Arco and Gutierrez-Rus, {Luis I.} and Risso, {Valeria A.} and Beatriz Ibarra-Molero and Yosuke Hoshino and Du{\v s}an Petrovi{\'c} and Jose Justicia and Cuerva, {Juan Manuel} and Adrian Romero-Rivera and Burckhard Seelig and Gavira, {Jose A.} and Kamerlin, {Shina C.L.} and Gaucher, {Eric A.} and Sanchez-Ruiz, {Jose M.}",

note = "Funding Information: This work was supported by Human Frontier Science Program Grant RGP0041 (J.M.S.-R., E.A.G., B.S., and S.C.L.K.), NIH grant R01AR069137 (E.A.G.), Department of Defense grant MURI W911NF-16-1-0372 (E.A.G.), the Swedish Research Council (2019-03499) (S.C.L.K.), the Knut and Alice Wallenberg Foundation (2018.0140 and 2019.0431) (S.C.L.K.), Spanish Ministry of Economy and Competitiveness/FEDER Funds Grants BIO2015-66426-R (J.M.S.-R.) RTI2018-097142-B-100 (J.M.S.-R.) and BIO2016-74875-P (J.A.G.). The simulations were enabled by resources provided by the Swedish National Infrastructure for Computing (SNIC) at UPPMAX partially funded by the Swedish Research Council through grant agreement no. 2016-07213. We acknowledge the Spanish Synchrotron Radiation Facility (ALBA, Barcelona) for the provision of synchrotron radiation facilities and the staff at XALOC beamline for their invaluable support. We are also grateful to Victoria Longobardo Polanco (Proteomic Unit, Institute of Parasitology and Biomedicine “L{\'o}pez-Neyra”) for help with mass spectrometry experiments and data analyses and to Juan Rom{\'a}n Luque Ortega (Molecular Interactions Facility, Centro de Investigaciones Biol{\'o}gicas Margarita Salas) for help with ultracentrifugation experiments and data analyses. Publisher Copyright: {\textcopyright} 2021, The Author(s).",

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doi = "10.1038/s41467-020-20630-1",

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T1 - Heme-binding enables allosteric modulation in an ancient TIM-barrel glycosidase

AU - Gamiz-Arco, Gloria

AU - Gutierrez-Rus, Luis I.

AU - Risso, Valeria A.

AU - Ibarra-Molero, Beatriz

AU - Hoshino, Yosuke

AU - Petrović, Dušan

AU - Justicia, Jose

AU - Cuerva, Juan Manuel

AU - Romero-Rivera, Adrian

AU - Seelig, Burckhard

AU - Gavira, Jose A.

AU - Kamerlin, Shina C.L.

AU - Gaucher, Eric A.

AU - Sanchez-Ruiz, Jose M.

N1 - Funding Information: This work was supported by Human Frontier Science Program Grant RGP0041 (J.M.S.-R., E.A.G., B.S., and S.C.L.K.), NIH grant R01AR069137 (E.A.G.), Department of Defense grant MURI W911NF-16-1-0372 (E.A.G.), the Swedish Research Council (2019-03499) (S.C.L.K.), the Knut and Alice Wallenberg Foundation (2018.0140 and 2019.0431) (S.C.L.K.), Spanish Ministry of Economy and Competitiveness/FEDER Funds Grants BIO2015-66426-R (J.M.S.-R.) RTI2018-097142-B-100 (J.M.S.-R.) and BIO2016-74875-P (J.A.G.). The simulations were enabled by resources provided by the Swedish National Infrastructure for Computing (SNIC) at UPPMAX partially funded by the Swedish Research Council through grant agreement no. 2016-07213. We acknowledge the Spanish Synchrotron Radiation Facility (ALBA, Barcelona) for the provision of synchrotron radiation facilities and the staff at XALOC beamline for their invaluable support. We are also grateful to Victoria Longobardo Polanco (Proteomic Unit, Institute of Parasitology and Biomedicine “López-Neyra”) for help with mass spectrometry experiments and data analyses and to Juan Román Luque Ortega (Molecular Interactions Facility, Centro de Investigaciones Biológicas Margarita Salas) for help with ultracentrifugation experiments and data analyses. Publisher Copyright: © 2021, The Author(s).

PY - 2021/12/1

Y1 - 2021/12/1

N2 - Glycosidases are phylogenetically widely distributed enzymes that are crucial for the cleavage of glycosidic bonds. Here, we present the exceptional properties of a putative ancestor of bacterial and eukaryotic family-1 glycosidases. The ancestral protein shares the TIM-barrel fold with its modern descendants but displays large regions with greatly enhanced conformational flexibility. Yet, the barrel core remains comparatively rigid and the ancestral glycosidase activity is stable, with an optimum temperature within the experimental range for thermophilic family-1 glycosidases. None of the ∼5500 reported crystallographic structures of ∼1400 modern glycosidases show a bound porphyrin. Remarkably, the ancestral glycosidase binds heme tightly and stoichiometrically at a well-defined buried site. Heme binding rigidifies this TIM-barrel and allosterically enhances catalysis. Our work demonstrates the capability of ancestral protein reconstructions to reveal valuable but unexpected biomolecular features when sampling distant sequence space. The potential of the ancestral glycosidase as a scaffold for custom catalysis and biosensor engineering is discussed.

AB - Glycosidases are phylogenetically widely distributed enzymes that are crucial for the cleavage of glycosidic bonds. Here, we present the exceptional properties of a putative ancestor of bacterial and eukaryotic family-1 glycosidases. The ancestral protein shares the TIM-barrel fold with its modern descendants but displays large regions with greatly enhanced conformational flexibility. Yet, the barrel core remains comparatively rigid and the ancestral glycosidase activity is stable, with an optimum temperature within the experimental range for thermophilic family-1 glycosidases. None of the ∼5500 reported crystallographic structures of ∼1400 modern glycosidases show a bound porphyrin. Remarkably, the ancestral glycosidase binds heme tightly and stoichiometrically at a well-defined buried site. Heme binding rigidifies this TIM-barrel and allosterically enhances catalysis. Our work demonstrates the capability of ancestral protein reconstructions to reveal valuable but unexpected biomolecular features when sampling distant sequence space. The potential of the ancestral glycosidase as a scaffold for custom catalysis and biosensor engineering is discussed.

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Heme-binding enables allosteric modulation in an ancient TIM-barrel glycosidase

Abstract

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