Structure and Mutation of Deoxypodophyllotoxin Synthase (DPS) from Podophyllum hexandrum

Zoe Ingold; Gideon James Grogan; Benjamin R. Lichman

doi:10.3389/fctls.2023.1178345

Structure and Mutation of Deoxypodophyllotoxin Synthase (DPS) from Podophyllum hexandrum

Zoe Ingold, Gideon James Grogan, Benjamin R. Lichman

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

Abstract

Deoxypodophyllotoxin synthase (DPS) is a 2-oxoglutarate (2-OG) dependent non-heme iron(II) dioxygenase that catalyzes the stereoselective ring-closing carbon-carbon bond formation of deoxypodophyllotoxin from the aryllignan (-)-yatein. Deoxypodophyllotoxin is a precursor of topoisomerase II inhibitors, which are on the World Health Organization’s list of essential medicines. Previous work has shown that DPS can accept a range of substrates, indicating it has potential in biocatalytic processes for the formation of diverse polycyclic aryllignans. Recent X-ray structures of the enzyme reveal possible roles for amino acid side chains in substrate recognition and mechanism, although a mutational analysis of DPS was not performed. Here, we present a structure of DPS at an improved resolution of 1.41 Å, in complex with the buffer molecule, Tris, coordinated to the active site iron atom. The structure has informed a mutational analysis of DPS, which suggests a role for a D224-K187 salt bridge in maintaining substrate interactions and a catalytic role for H165, perhaps as the base for the proton abstraction at the final rearomatization step. This work improves our understanding of specific residues’ contributions to the DPS mechanism and can inform future engineering of the enzyme mechanism and substrate scope for the development of a versatile biocatalyst.

Original language	English
Article number	1178345
Number of pages	10
Journal	Frontiers in Catalysis
Volume	3
DOIs	https://doi.org/10.3389/fctls.2023.1178345
Publication status	Published - 24 May 2023

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10.3389/fctls.2023.1178345Licence: CC BY

Structure and mutation of deoxypodophyllotoxin synthase (DPS) from Podophyllum hexandrum
© 2023 Ingold, Grogan and Lichman
Final published version, 1.3 MBLicence: CC BY

Cite this

@article{557334dba8334fda92f55b54eec9b75d,

title = "Structure and Mutation of Deoxypodophyllotoxin Synthase (DPS) from Podophyllum hexandrum",

abstract = "Deoxypodophyllotoxin synthase (DPS) is a 2-oxoglutarate (2-OG) dependent non-heme iron(II) dioxygenase that catalyzes the stereoselective ring-closing carbon-carbon bond formation of deoxypodophyllotoxin from the aryllignan (-)-yatein. Deoxypodophyllotoxin is a precursor of topoisomerase II inhibitors, which are on the World Health Organization{\textquoteright}s list of essential medicines. Previous work has shown that DPS can accept a range of substrates, indicating it has potential in biocatalytic processes for the formation of diverse polycyclic aryllignans. Recent X-ray structures of the enzyme reveal possible roles for amino acid side chains in substrate recognition and mechanism, although a mutational analysis of DPS was not performed. Here, we present a structure of DPS at an improved resolution of 1.41 {\AA}, in complex with the buffer molecule, Tris, coordinated to the active site iron atom. The structure has informed a mutational analysis of DPS, which suggests a role for a D224-K187 salt bridge in maintaining substrate interactions and a catalytic role for H165, perhaps as the base for the proton abstraction at the final rearomatization step. This work improves our understanding of specific residues{\textquoteright} contributions to the DPS mechanism and can inform future engineering of the enzyme mechanism and substrate scope for the development of a versatile biocatalyst.",

author = "Zoe Ingold and Grogan, {Gideon James} and Lichman, {Benjamin R.}",

note = "{\textcopyright} 2023 Ingold, Grogan and Lichman",

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doi = "10.3389/fctls.2023.1178345",

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T1 - Structure and Mutation of Deoxypodophyllotoxin Synthase (DPS) from Podophyllum hexandrum

AU - Ingold, Zoe

AU - Grogan, Gideon James

AU - Lichman, Benjamin R.

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Y1 - 2023/5/24

N2 - Deoxypodophyllotoxin synthase (DPS) is a 2-oxoglutarate (2-OG) dependent non-heme iron(II) dioxygenase that catalyzes the stereoselective ring-closing carbon-carbon bond formation of deoxypodophyllotoxin from the aryllignan (-)-yatein. Deoxypodophyllotoxin is a precursor of topoisomerase II inhibitors, which are on the World Health Organization’s list of essential medicines. Previous work has shown that DPS can accept a range of substrates, indicating it has potential in biocatalytic processes for the formation of diverse polycyclic aryllignans. Recent X-ray structures of the enzyme reveal possible roles for amino acid side chains in substrate recognition and mechanism, although a mutational analysis of DPS was not performed. Here, we present a structure of DPS at an improved resolution of 1.41 Å, in complex with the buffer molecule, Tris, coordinated to the active site iron atom. The structure has informed a mutational analysis of DPS, which suggests a role for a D224-K187 salt bridge in maintaining substrate interactions and a catalytic role for H165, perhaps as the base for the proton abstraction at the final rearomatization step. This work improves our understanding of specific residues’ contributions to the DPS mechanism and can inform future engineering of the enzyme mechanism and substrate scope for the development of a versatile biocatalyst.

AB - Deoxypodophyllotoxin synthase (DPS) is a 2-oxoglutarate (2-OG) dependent non-heme iron(II) dioxygenase that catalyzes the stereoselective ring-closing carbon-carbon bond formation of deoxypodophyllotoxin from the aryllignan (-)-yatein. Deoxypodophyllotoxin is a precursor of topoisomerase II inhibitors, which are on the World Health Organization’s list of essential medicines. Previous work has shown that DPS can accept a range of substrates, indicating it has potential in biocatalytic processes for the formation of diverse polycyclic aryllignans. Recent X-ray structures of the enzyme reveal possible roles for amino acid side chains in substrate recognition and mechanism, although a mutational analysis of DPS was not performed. Here, we present a structure of DPS at an improved resolution of 1.41 Å, in complex with the buffer molecule, Tris, coordinated to the active site iron atom. The structure has informed a mutational analysis of DPS, which suggests a role for a D224-K187 salt bridge in maintaining substrate interactions and a catalytic role for H165, perhaps as the base for the proton abstraction at the final rearomatization step. This work improves our understanding of specific residues’ contributions to the DPS mechanism and can inform future engineering of the enzyme mechanism and substrate scope for the development of a versatile biocatalyst.

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