Selective oxidative demethylation of veratric acid to vanillic acid by CYP199A4 from Rhodopseudomonas palustris HaA2

Bell, Stephen; Tan, Adrian B. H.; Johnson, Eachan O. D.; Wong, Luet‐Lok

doi:10.1039/b913487e

Cited by 68 publications

(165 citation statements)

References 35 publications

(45 reference statements)

Supporting

Mentioning

163

Contrasting

Order By: Relevance

“…The first electron transfer is rate limiting for a number of bacterial P450 systems including P450 cam (CYP101A1) from Pseudomonas putida (Brewer and Peterson, 1988), CYP199A2 and CYP199A4 from Rhodopseudomonas palustris (Bell et al, 2010b(Bell et al, , 2010c) and CYP101D1 from Novosphingobium aromaticivorans Bell et al, 2010a;Yang et al, 2010). It has been shown that the first flavin-to-heme electron transfer, though one of the slower steps in the overall catalytic cycle, is not rate limiting for palmitate and arachidonate oxidation by P450 BM3 (Munro et al, 1996;Ost et al, 2003;Whitehouse et al, 2011).…”

Section: Discussionmentioning

confidence: 99%

Chain length-dependent cooperativity in fatty acid binding and oxidation by cytochrome P450BM3 (CYP102A1)

et al. 2011

Self Cite

View full text Add to dashboard Cite

Fatty acid binding and oxidation kinetics for wild type P450 BM3 (CYP102A1) from Bacillus megaterium have been found to display chain length-dependent homotropic behavior. Laurate and 13-methyl-myristate display Michaelis-Menten behavior while there are slight deviations with myristate at low ionic strengths. Palmitate shows Michaelis-Menten kinetics and hyperbolic binding behavior in 100 mmol/L phosphate, pH 7.4, but sigmoidal kinetics (with an apparent intercept) in low ionic strength buffers and at physiological phosphate concentrations. In low ionic strength buffers both the heme domain and the full-length enzyme show complex palmitate binding behavior that indicates a minimum of four fatty acid binding sites, with high cooperativity for the binding of the fourth palmitate molecule, and the full-length enzyme showing tighter palmitate binding than the heme domain. The first flavin-to-heme electron transfer is faster for laurate, myristate and palmitate in 100 mmol/L phosphate than in 50 mmol/L Tris (pH 7.4), yet each substrate induces similar high-spin heme content. For palmitate in low phosphate buffer concentrations, the rate constant of the first electron transfer is much larger than k cat . The results suggest that phosphate has a specific effect in promoting the first electron transfer step, and that P450 BM3 could modulate Bacillus membrane morphology and fluidity via palmitate oxidation in response to the external phosphate concentration.

show abstract

Section: Discussionmentioning

confidence: 99%

Chain length-dependent cooperativity in fatty acid binding and oxidation by cytochrome P450BM3 (CYP102A1)

et al. 2011

Self Cite

View full text Add to dashboard Cite

show abstract

“…[1,15,16] For example, 4-ethylbenzoic acid is efficiently hydroxylated and desaturated by CYP199A4 to yield 4-(1-hydroxyethyl)-and 4-vinyl-benzoic acid, respectively. [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%

“…[14][15][16] For instance, CYP199A4 can bind and O-demethylate 2,4-and 3,4-dimethoxybenzoic acids with total regioselectivity at the 4-methoxy group to yield 2-methoxy-and 3-methoxy-4-hydroxybenzoic acids, respectively. [1,16] CYP199A4 has also…”

Section: Introductionmentioning

confidence: 99%

“…[6,7] The high enzymatic activity of CYP199A4 with this substrate is supported by a class I electron transfer chain consisting of a [2Fe-2S] ferredoxin (HaPux) and a flavin-dependent ferredoxin reductase (HaPuR) which mediate heme reduction by NADH. [1,[8][9][10] The stable, soluble nature and high activity of bacterial CYP enzymes, such as CYP199A4, results in them being of interest for applications in synthetic chemistry involving C-H bond oxidation. [11,12] Furthermore, these enzymes can be engineered via rational mutagenesis or directed evolution to enhance their activities and broaden their substrate range.…”

Section: Introductionmentioning

confidence: 99%

“…[1] CYP enzymes primarily catalyse the insertion of an oxygen atom from dioxygen into the carbon-hydrogen bonds of organic molecules. [2][3][4] Mechanistically, O-demethylation of 4-methoxybenzoic acid occurs via hydrogen abstraction by Compound I, [5] followed by oxygen rebound to give the hydroxylated hemiacetal which spontaneously decomposes to yield 4-hydroxybenzoic acid and formaldehyde.…”

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

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

Self Cite

View full text Add to dashboard Cite

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.

show abstract

Exploring the Factors which Result in Cytochrome P450 Catalyzed Desaturation Versus Hydroxylation

Coleman

Doherty

Zhang

et al. 2022

Chemistry An Asian Journal

Self Cite

View full text Add to dashboard Cite

The cytochrome P450 family of monooxygenase enzymes have essential biological roles involving the selective oxidation of carbon‐hydrogen bonds. They can also catalyze other important metabolic reactions including desaturation to form alkenes. Currently the factors that control the partition between P450 hydroxylation and desaturation pathways are poorly defined. The CYP199A4 enzyme from the bacterium Rhodopseudomonas palustris HaA2 catalyzes the oxidation of 4‐ethyl‐ and 4‐isopropyl‐ benzoic acids with hydroxylation and desaturation occurring in significant quantities. Here we demonstrate that 4‐cyclopropylbenzoic acid is regioselectively hydroxylated by CYP199A4 at the benzylic carbon. In contrast, the oxidation of 4‐n‐propylbenzoic acid by CYP199A4 results in three major metabolites: an alkene from desaturation and two hydroxylation products at the benzylic (Cα) and Cβ carbons in similar quantities. Extending the length of the alkyl substituent resulted in 4‐n‐butylbenzoic acid being oxidized at the benzylic position (45%) and desaturated (55%). In contrast, 4‐isobutylbenzoic generated very little alkene (5%) but was hydroxylated at the benzylic position (54%) and at the tertiary Cβ position (41%). The oxidation of 4‐n‐propylbenzoic acid by the F298 V mutant of CYP199A4 occurred with no hydroxylation at Cβ and a significant increase in metabolites arising from desaturation (73%). The X‐ray crystal structures of CYP199A4 with each substrate revealed that they bind in the active site with the alkyl substituent positioned over the heme. However, the longer alkylbenzoic acids were bound in a different conformation as was 4‐n‐propylbenzoic acid in the F298 V mutant. Overall, the changes in metabolite distribution could be ascribed to bond strength differences and the position of the alkyl group relative to the heme.

show abstract

Selective oxidative demethylation of veratric acid to vanillic acid by CYP199A4 from Rhodopseudomonas palustris HaA2

Cited by 68 publications

References 35 publications

Chain length-dependent cooperativity in fatty acid binding and oxidation by cytochrome P450BM3 (CYP102A1)

Chain length-dependent cooperativity in fatty acid binding and oxidation by cytochrome P450BM3 (CYP102A1)

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

Exploring the Factors which Result in Cytochrome P450 Catalyzed Desaturation Versus Hydroxylation

Contact Info

Product

Resources

About