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Regioselective oxidation of indole- and quinolinecarboxylic acids by cytochrome P450 CYP199A2

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Abstract

CYP199A2, a bacterial P450 monooxygenase from Rhodopseudomonas palustris, was previously reported to oxidize 2-naphthoic acid and 4-ethylbenzoic acid. In this study, we examined the substrate specificity and regioselectivity of CYP199A2 towards indole- and quinolinecarboxylic acids. The CYP199A2 gene was coexpressed with palustrisredoxin gene from R. palustris and putidaredoxin reductase gene from Pseudomonas putida to provide the redox partners of CYP199A2 in Escherichia coli. Following whole-cell assays, reaction products were identified by mass spectrometry and NMR spectroscopy. CYP199A2 did not exhibit any activity towards indole and indole-3-carboxylic acid, whereas this enzyme oxidized indole-2-carboxylic acid, indole-5-carboxylic acid, and indole-6-carboxylic acid. Indole-2-carboxylic acid was converted to 5- and 6-hydroxyindole-2-carboxylic acids at a ratio of 59:41. In contrast, the indole-6-carboxylic acid oxidation generated only one product, 2-indolinone-6-carboxylic acid, at a rate of 130 mol (mol P450)−1 min−1. Furthermore, CYP199A2 also oxidized quinoline-6-carboxylic acid, although this enzyme did not exhibit any activity towards quinoline and its derivatives with a carboxyl group at the C-2, C-3, or C-4 positions. The oxidation product of quinoline-6-carboxylic acid was identified to be 3-hydroxyquinoline-6-carboxylic acid, which was a novel compound. These results suggest that CYP199A2 may be a valuable biocatalyst for the regioselective oxidation of various aromatic carboxylic acids.

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Authors and Affiliations

  1. Department of Applied Chemistry, Faculty of Science and Engineering, Waseda University, 3-4-1 Ohkubo, Shinjuku-ku, Tokyo, 169-8555, Japan

    Toshiki Furuya & Kuniki Kino

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  1. Toshiki Furuya
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  2. Kuniki Kino
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Correspondence to Kuniki Kino.

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Table S1

NMR data for 5-hydroxyindole-2-carboxylic acid (DOC 35.5 kb)

Table S2

NMR data for 6-hydroxyindole-2-carboxylic acid (DOC 35.5 kb)

Table S3

NMR data for 2-indolinone-6-carboxylic acid (DOC 24.5 kb)

Table S4

NMR data for 3-hydroxyquinoline-6-carboxylic acid (DOC 27 kb)

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Furuya, T., Kino, K. Regioselective oxidation of indole- and quinolinecarboxylic acids by cytochrome P450 CYP199A2. Appl Microbiol Biotechnol 85, 1861–1868 (2010). https://doi.org/10.1007/s00253-009-2207-1

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