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

Chao, Rebecca R.; Lau, Ian C.-K.; Voss, James J. De; Bell, Stephen

doi:10.1002/cctc.201600951

Cited by 6 publications

(11 citation statements)

References 43 publications

(52 reference statements)

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“…It tolerates other substituents at the ortho and meta positions, whereas the carboxylate moiety is critical for substrate recognition . Interestingly, the single Ser244Asp mutation in the active site of the enzyme abolished the carboxylate dependence and extended the substrate scope to benzene derivatives …”

Section: Introductionmentioning

confidence: 99%

Exploring the Selective Demethylation of Aryl Methyl Ethers with a Pseudomonas Rieske Monooxygenase

et al. 2018

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Biocatalytic dealkylation of aryl methyl ethers is an attractive reaction for valorization of lignin components, as well as for deprotection of hydroxy functionalities in synthetic chemistry. We explored the demethylation of various aryl methyl ethers by using an oxidative demethylase from Pseudomonas sp. HR199. The Rieske monooxygenase VanA and its partner electron transfer protein VanB were recombinantly coexpressed in Escherichia coli and they constituted at least 25 % of the total protein content. Enzymatic transformations showed that VanB accepts NADH and NADPH as electron donors. The VanA–VanB system demethylates a number of aromatic substrates, the presence of a carboxylic acid moiety is essential, and the catalysis occurs selectively at the meta position to this carboxylic acid in the aromatic ring. The reaction is inhibited by the by‐product formaldehyde. Therefore, we tested three different cascade/tandem reactions for cofactor regeneration and formaldehyde elimination; in particular, conversion was improved by addition of formaldehyde dehydrogenase and formate dehydrogenase. Finally, the biocatalyst was applied for the preparation of protocatechuic acid from vanillic acid, giving a 77 % yield of the desired product. The described reaction may find application in the conversion of lignin components into diverse hydroxyaromatic building blocks and generally offers potential for new, mild methods for efficient unmasking of phenols.

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Section: Introductionmentioning

confidence: 99%

Exploring the Selective Demethylation of Aryl Methyl Ethers with a Pseudomonas Rieske Monooxygenase

et al. 2018

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“…Improvements of up to 900-fold in the rate of substrate oxidation were observed for the S244D mutant with 4-methoxyphenol. [15] While the CYP199A4 S244D variant resulted in improved activity for non-acidic benzene substrates, there was little or no increase in the binding affinity. [15] Here we first investigate the oxidation of methyl and ethyl substituted benzene derivatives with the S244D mutant of CYP199A4 to determine if regioselective hydroxylation at the Figure 1.…”

Section: Introductionmentioning

confidence: 99%

“…[15] While the CYP199A4 S244D variant resulted in improved activity for non-acidic benzene substrates, there was little or no increase in the binding affinity. [15] Here we first investigate the oxidation of methyl and ethyl substituted benzene derivatives with the S244D mutant of CYP199A4 to determine if regioselective hydroxylation at the Figure 1. The active site of CYP199A4 with 4-methoxybenzoic acid bound (PDB: 4DO1).…”

Section: Introductionmentioning

confidence: 99%

“…[9b,16] The mutation of serine 244 of CYP199A4 to an aspartate residue (S244D), and the equivalent mutation in CYP199A2, significantly improved the activity of both enzymes towards non-acidic benzene substrates. [15,17] The WT CYP199A4 enzyme demethylates 4-methoxybenzoic acid at a significantly higher rate than the S244D variant but the reverse is true when the carboxylate moiety is changed to a different functional group, such as an aldehyde or alcohol. For example, the product formation rate with WT CYP199A4 and 4-methoxybenzaldehyde is 3.0 nmol (nmol-CYP) À 1 min À 1 , while for the S244D variant it is 860 nmol (nmol-CYP) À 1 min À 1 .…”

Section: Introductionmentioning

confidence: 99%

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The Stereoselective Oxidation of para‐Substituted Benzenes by a Cytochrome P450 Biocatalyst

Chao

Lau

Coleman

et al. 2021

Chemistry A European J

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The serine 244 to aspartate (S244D) variant of the cytochrome P450 enzyme CYP199A4 was used to expand its substrate range beyond benzoic acids. Substrates, in which the carboxylate group of the benzoic acid moiety is replaced were oxidised with high activity by the S244D mutant (product formation rates > 60 nmol.(nmol-CYP) À 1 .min À 1 ) and with total turnover numbers of up to 20,000. Ethyl αhydroxylation was more rapid than methyl oxidation, styrene epoxidation and S-oxidation. The S244D mutant catalysed the ethyl hydroxylation, epoxidation and sulfoxidation reactions with an excess of one stereoisomer (in some instances up to > 98 %). The crystal structure of 4-methoxybenzoic acidbound CYP199A4 S244D showed that the active site architecture and the substrate orientation were similar to that of the WT enzyme. Overall, this work demonstrates that CYP199A4 can catalyse the stereoselective hydroxylation, epoxidation or sulfoxidation of substituted benzene substrates under mild conditions resulting in more sustainable transformations using this heme monooxygenase enzyme.

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“…Some wild-type organisms have found applications in drug synthesis. 9 The solubility and ease of high level heterologous production of bacterial CYPs in Escherichia coli make them attractive for developing new biotechnological applications in, for example, the synthesis of fine chemicals, [10][11][12][13][14][15][16][17][18][19] intermediates, 14, [20][21][22] drugs, [23][24][25][26][27][28] and antibiotics. [29][30][31] However, many of the Class I CYP enzymes are not genetically associated with their Fdx.…”

Section: Introductionmentioning

confidence: 99%

A Structural Model of a P450-Ferredoxin Complex from Orientation-Selective Double Electron–Electron Resonance Spectroscopy

et al. 2018

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Cytochrome P450 (CYP) monooxygenases catalyze the oxidation of chemically inert carbon-hydrogen bonds in diverse endogenous and exogenous organic compounds by atmospheric oxygen. This C-H bond oxy-functionalization activity has huge potential in biotechnological applications. Class I CYPs receive the two electrons required for oxygen activation from NAD(P)H via a ferredoxin reductase and ferredoxin. The interaction of Class I CYPs with their cognate ferredoxin is specific. In order to reconstitute the activity of diverse CYPs, structural characterization of CYP-ferredoxin complexes is necessary, but little structural information is available. Here we report a structural model of such a complex (CYP199A2-HaPux) in frozen solution derived from distance and orientation restraints gathered by the EPR technique of orientation-selective double electron-electron resonance (os-DEER). The long-lived oscillations in the os-DEER spectra were well modeled by a single orientation of the CYP199A2-HaPux complex. The structure is different from the two known Class I CYP-Fdx structures: CYP11A1-Adx and CYP101A1-Pdx. At the protein interface, HaPux residues in the [FeS] cluster-binding loop and the α3 helix and the C-terminus residue interact with CYP199A2 residues in the proximal loop and the C helix. These residue contacts are consistent with biochemical data on CYP199A2-ferredoxin binding and electron transfer. Electron-tunneling calculations indicate an efficient electron-transfer pathway from the [FeS] cluster to the heme. This new structural model of a CYP-Fdx complex provides the basis for tailoring CYP enzymes for which the cognate ferredoxin is not known, to accept electrons from HaPux and display monooxygenase activity.

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

Cited by 6 publications

References 43 publications

Exploring the Selective Demethylation of Aryl Methyl Ethers with a Pseudomonas Rieske Monooxygenase

Exploring the Selective Demethylation of Aryl Methyl Ethers with a Pseudomonas Rieske Monooxygenase

The Stereoselective Oxidation of para‐Substituted Benzenes by a Cytochrome P450 Biocatalyst

A Structural Model of a P450-Ferredoxin Complex from Orientation-Selective Double Electron–Electron Resonance Spectroscopy

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