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

Furuya, Toshiki; Kino, Kuniki

doi:10.1007/s00253-009-2207-1

Cited by 27 publications

(21 citation statements)

References 24 publications

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“…We explored the catalytic potential of CYP199A2 for the oxidation of coumaric and cinnamic acids. Whole-cell assays were performed using E. coli cells that coexpressed CYP199A2 with putidaredoxin reductase from P. putida and palustrisredoxin from R. palustris (12). HPLC analysis of the reaction of CYP199A2 with p-coumaric acid showed a major peak (retention time, 4.2 min) in addition to the substrate peak (5.7 min) (see Fig.…”

Section: Resultsmentioning

confidence: 99%

“…The wild-type or mutant CYP199A2 gene was coexpressed with the putidaredoxin reductase gene (pdR) from Pseudomonas putida (30) and the palustrisredoxin gene (pux) from R. palustris (2) to provide the redox partners of CYP199A2 in Escherichia coli. pET21a, carrying the wild-type or mutant CYP199A2 gene, and pMW218, carrying the pdR and pux genes, which was previously constructed (12), were simultaneously introduced into E. coli BL21(DE3) cells (Novagen). These recombinant E. coli cells were cultivated in LuriaBertani medium (1% Bacto tryptone, 0.5% Bacto yeast extract, 1% NaCl [pH 7.0]) supplemented with ampicillin (100 g/ml) and kanamycin (100 g/ml) at 25°C.…”

Section: Methodsmentioning

confidence: 99%

“…CYP199A2 was recently discovered in Rhodopseudomonas palustris and has been shown to exhibit oxidation activity for aromatic carboxylic acids, including 2-naphthoic acid, 4-ethylbenzoic acid, and indole-and quinolinecarboxylic acids (3,11,12,13). We also subjected CYP199A2 to site-directed mutagenesis based on its crystal structure to create mutants with novel and improved catalytic properties.…”

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Biotechnological Production of Caffeic Acid by Bacterial Cytochrome P450 CYP199A2

Furuya

Arai

Kino

2012

Appl Environ Microbiol

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ABSTRACTCaffeic acid is a biologically active molecule that has various beneficial properties, including antioxidant, anticancer, and anti-inflammatory activities. In this study, we explored the catalytic potential of a bacterial cytochrome P450, CYP199A2, for the biotechnological production of caffeic acid. When the CYP199A2 enzyme was reacted withp-coumaric acid, it stoichiometrically produced caffeic acid. The crystal structure of CYP199A2 shows that Phe at position 185 is situated directly above, and only 6.35 Å from, the heme iron. This F185 residue was replaced with hydrophobic or hydroxylated amino acids using site-directed mutagenesis to create mutants with novel and improved catalytic properties. In whole-cell assays with the known substrate of CYP199A2, 2-naphthoic acid, only the wild-type enzyme hydroxylated 2-naphthoic acid at the C-7 and C-8 positions, whereas all of the active F185 mutants exhibited a preference for C-5 hydroxylation. Interestingly, several F185 mutants (F185V, F185L, F185I, F185G, and F185A mutants) also acquired the ability to hydroxylate cinnamic acid, which was not hydroxylated by the wild-type enzyme. These results demonstrate that F185 is an important residue that controls the regioselectivity and the substrate specificity of CYP199A2. Furthermore,Escherichia colicells expressing the F185L mutant exhibited 5.5 times higher hydroxylation activity forp-coumaric acid than those expressing the wild-type enzyme. By using the F185L whole-cell catalyst, the production of caffeic acid reached 15 mM (2.8 g/liter), which is the highest level so far attained in biotechnological production of this compound.

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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confidence: 99%

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Biotechnological Production of Caffeic Acid by Bacterial Cytochrome P450 CYP199A2

Furuya

Arai

Kino

2012

Appl Environ Microbiol

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“…[1] The closely related CYP199A2 enzyme, from the CGA009 strain of R. palustris, has been reported to be a biocatalyst for the oxyfunctionalisation of 4-methoxybenzoic acid and various other aromatic carboxylic acids including 2-naphthoic, indole-6-carboxylic and cinnamic acids. [7,[17][18][19][20] Recently, the wild-type enzyme and mutant forms of CYP199A2…”

Section: Accepted M Manuscriptmentioning

confidence: 99%

“…[1,17,19,21,32] In order to investigate the whole-cell turnover of these substrates, we utilised a system developed in our laboratory which produced CYP199A4, HaPux and HaPuR in E. coli. [1] Using low cell density cultures (3 g cell wet weight.L 1 ), we have shown that 4-methoxybenzoic acid was rapidly converted to 4-hydroxybenzoic acid with 2 mM substrate being completely turned over in less than 4 hours and 4 mM in 24 hours (Fig.…”

Section: Whole-cell Oxidation Turnover Assaysmentioning

confidence: 99%

The importance of the benzoic acid carboxylate moiety for substrate recognition by CYP199A4 from Rhodopseudomonas palustris HaA2

Coleman

Chao

Voss

et al. 2016

Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics

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The importance of the benzoic acid carboxylate moiety for substrate recognition by CYP199A4 from Rhodopseudomonas palustris HaA2, BBA -Proteins and Proteomics (2016), doi: 10.1016/j.bbapap.2016 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. Abstract BackgroundThe cytochrome P450 enzyme CYP199A4 can efficiently demethylate 4-methoxybenzoic acid. The substrate is positioned in the enzyme active site with the methoxy group ideally positioned for demethylation. This occurs through interactions of hydrophobic benzene ring with aromatic phenylalanine residues and the charged carboxylate group with polar and basic amino acids. MethodsIn vitro substrate binding and kinetic turnovers assays coupled with HPLC and GC-MS analysis and whole-cell oxidation turnovers. ResultsModification of the carboxylate group to an amide or aldehyde resulted in substrate binding, as judged by the almost total shift of the spin state to the high-spin form, but binding was three orders of magnitude weaker. Changing the carboxylate to phenol alcohol, ketone, ester and nitro groups and boronic, sulfinic and sulfonic acids resulted in a dramatic reduction in the binding affinity. Even phenylacetic acids were mediocre substrates for CYP199A4, despite maintaining a carboxylate group. The weaker binding of all of these substrates results in lower levels of turnover activity and product formation compared to 4-methoxybenzoic acid. ConclusionSubstrate binding to CYP199A4 is tightly regulated by interactions between the 4-methoxybenzoic acid and the amino acids in the active site. The benzoic acid carboxylate moiety is critical for optimal substrate binding and turnover activity with CYP199A4. General SignificanceAn understanding of how the CYP199A4 enzyme has evolved to be highly selective for para-substituted benzoic acids. This provides valuable insight into how other, as yet structurally uncharacterised, monooxygenase enzymes may bind benzoic acid substrates. A C C E P T E D M A N U S C R I P T ACCEPTED MANUSCRIPT3 Highlights CYP199A4 has high selectivity for the carboxylate group of 4-methoxybenzoic acid.This selectivity arises from interactions with arginine and serine residues.Alternative functional groups can bind to CYP199A4 but with low affinity.The activities of the in vitro enzyme assays can be extrapolated to whole-cell oxidation systems.The high regioselectivity for oxidation at the para-substituent is maintained.

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Modification of an Enzyme Biocatalyst for the Efficient and Selective Oxidative Demethylation of para‐Substituted Benzene Derivatives

et al. 2016

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The bacterial CYP199A4 enzyme is able to oxidise a narrow range of aromatic acids, which includes 4‐methoxybenzoic acid, efficiently. A serine 244 to aspartate variant was identified with enhanced activity for a wide range of para‐methoxy‐substituted benzenes. Substrates in which the acidic benzoic acid moiety is replaced with a phenol and the amide, aldehyde and bromide analogues were all oxidised with high activity by the S244D mutant (product formation rate >600 nmol nmolCYP−1 min−1) with turnover numbers of up to 20 000. If the carboxylate moiety was modified to a nitro, ketone, boronic acid, hydroxymethyl or nitrile group, these substrates were also oxidised at a significantly higher activity by S244D than the wild‐type enzyme. 3,4‐Dimethoxybenzaldehyde was demethylated selectively and oxidatively to 3‐methoxy‐4‐hydroxybenzaldehyde by the S244D mutant 84‐fold more rapidly than with the wild‐type enzyme. CYP199A4 would have applications in the catalytic regioselective oxidative demethylation of suitably substituted benzene substrates under mild conditions and in the presence of more oxidatively sensitive functional groups, such as an aldehyde.

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

Cited by 27 publications

References 24 publications

Biotechnological Production of Caffeic Acid by Bacterial Cytochrome P450 CYP199A2

Biotechnological Production of Caffeic Acid by Bacterial Cytochrome P450 CYP199A2

The importance of the benzoic acid carboxylate moiety for substrate recognition by CYP199A4 from Rhodopseudomonas palustris HaA2

Modification of an Enzyme Biocatalyst for the Efficient and Selective Oxidative Demethylation of para‐Substituted Benzene Derivatives

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