Biotechnological Production of Caffeic Acid by Bacterial Cytochrome P450 CYP199A2

Furuya, Toshiki; Arai, Yuka; Kino, Kuniki

doi:10.1128/aem.01103-12

Cited by 59 publications

(52 citation statements)

References 41 publications

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“…Additionally, riboflavin (1 mM) was added for enhanced metabolism, 5-aminolevulinic acid (1 mM) for enhanced heme synthesis, and FeCl 3 (0.25 mM) for increased CYP and Fd functionality (i.e. supplying iron for the CYP heme and Fd) 56 . Substrates were incubated for three days in the cultures while shaken at 30 °C and 225 RPM.…”

Section: Methodsmentioning

confidence: 99%

Elucidation of gibberellin biosynthesis in bacteria reveals convergent evolution

et al. 2016

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Gibberellins (GAs) are crucial phytohormones involved in many aspects of plant growth and development, including plant-microbe interactions, which has led to GA production by plant-associated fungi and bacteria as well. While the GA biosynthetic pathways in plants and fungi have been elucidated and found to have independently arisen through convergent evolution, little has been uncovered about GA biosynthesis in bacteria. Some nitrogen-fixing/symbiotic, legume-associated rhizobia, including Bradyrhizobium japonicum, the symbiont of soybean, and Sinorhizobium fredii, a broad-host-nodulating species, contain a putative GA biosynthetic operon/gene cluster. Through functional characterization of five unknown genes, we demonstrate that this operon encodes the enzymes necessary to produce GA9, thereby elucidating bacterial GA biosynthesis. The distinct nature of these enzymes indicates that bacteria have independently evolved a third biosynthetic pathway for GA production. Furthermore, our results also reveal a central biochemical logic that is followed in all three convergently evolved GA biosynthetic pathways.

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Section: Methodsmentioning

confidence: 99%

Elucidation of gibberellin biosynthesis in bacteria reveals convergent evolution

et al. 2016

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“…[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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“…In the last years, several studies have used the enzymes involved in the phenylpropanoid pathway to produce compounds other than curcuminoids, such as phenylpropanoic acids (coumaric acid, caffeic acid, and ferulic acid) (140,141,151,152,(154)(155)(156)(157), stilbenoids (155,158), and flavonoids (116,117,150,155,(159)(160)(161)(162). In the design and construction of those pathways, some enzymes from other organisms were used that could also be considered for the optimization of the heterologous production of curcuminoids.…”

Section: Figmentioning

confidence: 99%

“…Furuya et al (156) found that Rhodopseudomonas palustris cytochrome P450 CYP199A2 possesses hydroxylation activity toward p-coumaric acid (and cinnamic acid) and subjected it to site-directed mutagenesis to create mutants with novel and improved catalytic properties. E. coli coexpressed CYP199A2 with the redox partners' putidaredoxin reductase (pdr) from Pseudomonas putida and palustrisredoxin (pux) from R. palustris.…”

Section: Figmentioning

confidence: 99%

Heterologous Production of Curcuminoids

Rodrigues

Prather

Kluskens

et al. 2015

Microbiol Mol Biol Rev

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SUMMARY Curcuminoids, components of the rhizome of turmeric, show several beneficial biological activities, including anticarcinogenic, antioxidant, anti-inflammatory, and antitumor activities. Despite their numerous pharmaceutically important properties, the low natural abundance of curcuminoids represents a major drawback for their use as therapeutic agents. Therefore, they represent attractive targets for heterologous production and metabolic engineering. The understanding of biosynthesis of curcuminoids in turmeric made remarkable advances in the last decade, and as a result, several efforts to produce them in heterologous organisms have been reported. The artificial biosynthetic pathway (e.g., in Escherichia coli ) can start with the supplementation of the amino acid tyrosine or phenylalanine or of carboxylic acids and lead to the production of several natural curcuminoids. Unnatural carboxylic acids can also be supplemented as precursors and lead to the production of unnatural compounds with possibly novel therapeutic properties. In this paper, we review the natural conversion of curcuminoids in turmeric and their production by E. coli using an artificial biosynthetic pathway. We also explore the potential of other enzymes discovered recently or already used in other similar biosynthetic pathways, such as flavonoids and stilbenoids, to increase curcuminoid yield and activity.

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Biotechnological Production of Caffeic Acid by Bacterial Cytochrome P450 CYP199A2

Cited by 59 publications

References 41 publications

Elucidation of gibberellin biosynthesis in bacteria reveals convergent evolution

Elucidation of gibberellin biosynthesis in bacteria reveals convergent evolution

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

Heterologous Production of Curcuminoids

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