Expanding the Substrate Scope of Nitrating Cytochrome P450 TxtE by Active Site Engineering of a Reductase Fusion

Rakesh Saroay; Gheorghe Doru Roiban; Lona M. Alkhalaf; Gregory L. Challis

doi:10.1002/cbic.202100145

Expanding the Substrate Scope of Nitrating Cytochrome P450 TxtE by Active Site Engineering of a Reductase Fusion

Rakesh Saroay, Gheorghe Doru Roiban, Lona M. Alkhalaf, Gregory L. Challis

Biochemistry & Molecular Biology

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

12 Citations (Scopus)

Abstract

Aromatic nitration reactions are a cornerstone of organic chemistry, but are challenging to scale due to corrosive reagents and elevated temperatures. The cytochrome P450 TxtE nitrates the indole 4-position of l-tryptophan at room temperature using NO, O₂ and NADPH, and has potential to be developed into a useful aromatic nitration biocatalyst. However, its narrow substrate scope (requiring both the α-amino acid and indole functionalities) have hindered this. Screening of an R59 mutant library of a TxtE-reductase fusion protein identified a variant (R59C) that nitrates tryptamine, which is not accepted by native TxtE. This variant exhibits a broader substrate scope than the wild type enzyme and is able to nitrate a range of tryptamine analogues, with significant alterations to the aromatic and aminoethyl moieties.

Original language	English
Pages (from-to)	2262-2265
Number of pages	4
Journal	ChemBioChem
Volume	22
Issue number	13
DOIs	https://doi.org/10.1002/cbic.202100145
Publication status	Published - 1 Jul 2021

Keywords

Biocatalysis
Indole
Nitration
Protein Engineering

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10.1002/cbic.202100145Licence: CC BY

Cite this

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title = "Expanding the Substrate Scope of Nitrating Cytochrome P450 TxtE by Active Site Engineering of a Reductase Fusion",

abstract = "Aromatic nitration reactions are a cornerstone of organic chemistry, but are challenging to scale due to corrosive reagents and elevated temperatures. The cytochrome P450 TxtE nitrates the indole 4-position of l-tryptophan at room temperature using NO, O2 and NADPH, and has potential to be developed into a useful aromatic nitration biocatalyst. However, its narrow substrate scope (requiring both the α-amino acid and indole functionalities) have hindered this. Screening of an R59 mutant library of a TxtE-reductase fusion protein identified a variant (R59C) that nitrates tryptamine, which is not accepted by native TxtE. This variant exhibits a broader substrate scope than the wild type enzyme and is able to nitrate a range of tryptamine analogues, with significant alterations to the aromatic and aminoethyl moieties.",

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author = "Rakesh Saroay and Roiban, {Gheorghe Doru} and Alkhalaf, {Lona M.} and Challis, {Gregory L.}",

note = "Funding Information: This research was supported by grants from the BBSRC (Grant Reference BB/L502017/1) and GlaxoSmithKline to G.L.C. The Dionex 3000RS/Bruker MaXis Impact instrument used in this work was purchased with a grant to G.L.C. from the BBSRC (BB/K002341/1). Funding Information: This research was supported by grants from the BBSRC (Grant Reference BB/L502017/1) and GlaxoSmithKline to G.L.C. The Dionex 3000RS/Bruker MaXis Impact instrument used in this work was purchased with a grant to G.L.C. from the BBSRC (BB/K002341/1). Publisher Copyright: {\textcopyright} 2021 The Authors. ChemBioChem published by Wiley-VCH GmbH Copyright: Copyright 2021 Elsevier B.V., All rights reserved.",

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AU - Challis, Gregory L.

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N2 - Aromatic nitration reactions are a cornerstone of organic chemistry, but are challenging to scale due to corrosive reagents and elevated temperatures. The cytochrome P450 TxtE nitrates the indole 4-position of l-tryptophan at room temperature using NO, O2 and NADPH, and has potential to be developed into a useful aromatic nitration biocatalyst. However, its narrow substrate scope (requiring both the α-amino acid and indole functionalities) have hindered this. Screening of an R59 mutant library of a TxtE-reductase fusion protein identified a variant (R59C) that nitrates tryptamine, which is not accepted by native TxtE. This variant exhibits a broader substrate scope than the wild type enzyme and is able to nitrate a range of tryptamine analogues, with significant alterations to the aromatic and aminoethyl moieties.

AB - Aromatic nitration reactions are a cornerstone of organic chemistry, but are challenging to scale due to corrosive reagents and elevated temperatures. The cytochrome P450 TxtE nitrates the indole 4-position of l-tryptophan at room temperature using NO, O2 and NADPH, and has potential to be developed into a useful aromatic nitration biocatalyst. However, its narrow substrate scope (requiring both the α-amino acid and indole functionalities) have hindered this. Screening of an R59 mutant library of a TxtE-reductase fusion protein identified a variant (R59C) that nitrates tryptamine, which is not accepted by native TxtE. This variant exhibits a broader substrate scope than the wild type enzyme and is able to nitrate a range of tryptamine analogues, with significant alterations to the aromatic and aminoethyl moieties.

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Expanding the Substrate Scope of Nitrating Cytochrome P450 TxtE by Active Site Engineering of a Reductase Fusion

Abstract

Keywords

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ARC Centre of Excellence for Innovations in Peptide and Protein Science

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Expanding the Substrate Scope of Nitrating Cytochrome P450 TxtE by Active Site Engineering of a Reductase Fusion

Abstract

Keywords

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ARC Centre of Excellence for Innovations in Peptide and Protein Science

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