Expanding the Substrate Scope of Nitrating Cytochrome P450 TxtE by Active Site Engineering of a Reductase Fusion |
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Authors: | Dr Rakesh Saroay Dr Gheorghe-Doru Roiban Dr Lona M Alkhalaf Prof Gregory L Challis |
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Affiliation: | 1. Department of Chemistry, University of Warwick, Coventry, CV4 7AL UK;2. Synthetic Biochemistry, Medicinal Science & Technology, GlaxoSmithKline, Medicines Research Centre, Gunnels Wood Road, Stevenage, SG1 2NY UK |
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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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Keywords: | Biocatalysis Indole Nitration Protein Engineering |
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