Coenzyme A‐Conjugated Cinnamic Acids – Enzymatic Synthesis of a CoA‐Ester Library and Application in Biocatalytic Cascades to Vanillin Derivatives

Dippe, Martin; Bauer, Anne‐Katrin; Porzel, Andrea; Funke, Evelyn; Müller, Anna Ophelia; Schmidt, Jürgen; Beier, Maria; Wessjohann, Ludger A.

doi:10.1002/adsc.201900892

Cited by 14 publications

(17 citation statements)

References 24 publications

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“…The active site volume of Nt 4CL2 was not only comparable with Hc AAE1 (relative volume 1.02/1) but also to the promiscuous A. thaliana 4CL2 ( At 4CL2; 1.03/1, Dippe et al ., 2019). Consequently, docking experiments of At 4CL2 showed that the promiscuity of the enzyme was defined by the amino acid composition rather than the size of the active site (Dippe et al ., 2019), ruling out a dominant role of the size exclusion mechanism for the recognition of the substrates. In the Hc AAE1 SBP, Phe‐397 appears to form a π–π stacking interaction with the phenyl ring of benzoic acid.…”

Section: Discussionmentioning

confidence: 99%

A promiscuous coenzyme A ligase provides benzoyl‐coenzyme A for xanthone biosynthesis in Hypericum

et al. 2020

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Benzoic acid-derived compounds, such as polyprenylated benzophenones and xanthones, attract the interest of scientists due to challenging chemical structures and diverse biological activities. The genus Hypericum is of high medicinal value, as exemplified by H. perforatum. It is rich in benzophenone and xanthone derivatives, the biosynthesis of which requires the catalytic activity of benzoate-coenzyme A (benzoate-CoA) ligase (BZL), which activates benzoic acid to benzoyl-CoA. Despite remarkable research so far done on benzoic acid biosynthesis in planta, all previous structural studies of BZL genes and proteins are exclusively related to benzoate-degrading microorganisms. Here, a transcript for a plant acyl-activating enzyme (AAE) was cloned from xanthone-producing Hypericum calycinum cell cultures using transcriptomic resources. An increase in the HcAAE1 transcript level preceded xanthone accumulation after elicitor treatment, as previously observed with other pathway-related genes. Subcellular localization of reporter fusions revealed the dual localization of HcAAE1 to cytosol and peroxisomes owing to a type 2 peroxisomal targeting signal. This result suggests the generation of benzoyl-CoA in Hypericum by the CoA-dependent non-b-oxidative route. A luciferase-based substrate specificity assay and the kinetic characterization indicated that HcAAE1 exhibits promiscuous substrate preference, with benzoic acid being the sole aromatic substrate accepted. Unlike 4coumarate-CoA ligase and cinnamate-CoA ligase enzymes, HcAAE1 did not accept 4-coumaric and cinnamic acids, respectively. The substrate preference was corroborated by in silico modeling, which indicated valid docking of both benzoic acid and its adenosine monophosphate intermediate in the HcAAE1/BZL active site cavity.

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Section: Discussionmentioning

confidence: 99%

A promiscuous coenzyme A ligase provides benzoyl‐coenzyme A for xanthone biosynthesis in Hypericum

et al. 2020

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“…Enzyme variants were constructed by mutagenesis of pET28a(+)‐4HPA3H by the Quik Change II Site‐Directed Mutagenesis Kit (Agilent, Santa Clara, USA). The recombinant enzymes, which carried a hexahistidine tag at their N‐terminal, were produced in E. coli BL21(DE3) (see Supporting Information) and purified by immobilized Co 2+ ion affinity chromatography as described previously [39] . Purified proteins were checked for homogeneity by sodium dodecyl sulfate polyacrylamide gel electrophoresis, and their concentration was determined by the method of Bradford [40]…”

Section: Methodsmentioning

confidence: 99%

“…The recombinant enzymes, which carried a hexahistidine tag at their N-terminal, were produced in E. coli BL21(DE3) (see Supporting Information) and purified by immobilized Co 2 + ion affinity chromatography as described previously. [39] Purified proteins were checked for homogeneity by sodium dodecyl sulfate polyacrylamide gel electrophoresis, and their concentration was determined by the method of Bradford. [40] Whole-cell bioconversions and product identification Reactions (10 mL) were performed in duplicates in auto-inducing medium [41] containing 200 μM hydroxylase substrate.…”

Section: Experimental Section Enzyme Production and Quantificationmentioning

confidence: 99%

Engineered Bacterial Flavin‐Dependent Monooxygenases for the Regiospecific Hydroxylation of Polycyclic Phenols

et al. 2022

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4-Hydroxyphenylacetate 3-hydroxylase (4HPA3H), a flavin-dependent monooxygenase from E. coli that catalyzes the hydroxylation of monophenols to catechols, was modified by rational redesign to convert also more bulky substrates, especially phenolic natural products like phenylpropanoids, flavones or coumarins. Selected amino acid positions in the binding pocket of 4HPA3H were exchanged with residues from the homologous protein from Pseudomonas aeruginosa, yielding variants with improved conversion of spacious substrates such as the flavonoid naringenin or the alkaloid mimetic 2-hydroxycarbazole. Reactions were followed by an adapted Fe(III)-catechol chromogenic assay selective for the products. Especially substitution of the residue Y301 facilitated modulation of substrate specificity: introduction of nonaromatic but hydrophobic (iso)leucine resulted in the preference of the substrate ferulic acid (having a guaiacyl (guajacyl) moiety, part of the vanilloid motif) over unsubstituted monophenols. The in vivo (whole-cell biocatalysts) and in vitro (three-enzyme cascade) transformations of substrates by 4HPA3H and its optimized variants was strictly regiospecific and proceeded without generation of byproducts.

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“…The CoA-dependent pathway can be described as follows: feruloyl–CoA synthetase (FCS) ligates the acrylic group of phenylpropanoid acids and CoA to form acyl–CoA by adenosine triphosphate (ATP) driving. Then the enoyl–CoA hydratase (ECH) converts acyl–CoA into an aldehyde group . FCS is the rate-limiting enzyme throughout the progress of aromatic aldehyde synthesis. − Our previous study elevated vanillin production by enhancing the expression level of FCS .…”

Section: Introductionmentioning

confidence: 97%

“…Simply regulating the expression level of FCS could not solve the low aromatic aldehydes production caused by rate-limited FCS. Hence, the poor catalytic ability of wild-type FCS to different natural phenylpropanoid acids has become a bottleneck for the efficient biosynthesis of aromatic aldehydes. , …”

Section: Introductionmentioning

confidence: 99%

Engineering a Feruloyl–Coenzyme A Synthase for Bioconversion of Phenylpropanoid Acids into High-Value Aromatic Aldehydes

Chen

Jiang

Kang³

et al. 2022

J. Agric. Food Chem.

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Aromatic aldehydes find extensive applications in food, perfume, pharmaceutical, and chemical industries. However, a limited natural enzyme selectivity has become the bottleneck of bioconversion of aromatic aldehydes from natural phenylpropanoid acids. Here, based on the original structure of feruloyl–coenzyme A (CoA) synthetase (FCS) from Streptomyces sp. V-1, we engineered five substrate-binding domains to match specific phenylpropanoid acids. FcsCIAE407A/K483L, FcsMAE407R/I481R/K483R, FcsHAE407K/I481K/K483I, FcsCAE407R/I481R/K483T, and FcsFAE407R/I481K/K483R showed 9.96-, 10.58-, 4.25-, 6.49-, and 8.71-fold enhanced catalytic efficiency for degrading CoA thioesters of cinnamic acid, 4-methoxycinnamic acid, 4-hydroxycinnamic acid, caffeic acid, and ferulic acid, respectively. Molecular dynamics simulation illustrated that novel substrate-binding domains formed strong interaction forces with substrates’ methoxy/hydroxyl group and provided hydrophobic/alkaline catalytic surfaces. Five recombinant E. coli with FCS mutants were constructed with the maximum benzaldehyde, p-anisaldehyde, p-hydroxybenzaldehyde, protocatechualdehyde, and vanillin productivity of 6.2 ± 0.3, 5.1 ± 0.23, 4.1 ± 0.25, 7.1 ± 0.3, and 8.7 ± 0.2 mM/h, respectively. Hence, our study provided novel and efficient enzymes for the bioconversion of phenylpropanoid acids into aromatic aldehydes.

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Coenzyme A‐Conjugated Cinnamic Acids – Enzymatic Synthesis of a CoA‐Ester Library and Application in Biocatalytic Cascades to Vanillin Derivatives

Cited by 14 publications

References 24 publications

A promiscuous coenzyme A ligase provides benzoyl‐coenzyme A for xanthone biosynthesis in Hypericum

A promiscuous coenzyme A ligase provides benzoyl‐coenzyme A for xanthone biosynthesis in Hypericum

Engineered Bacterial Flavin‐Dependent Monooxygenases for the Regiospecific Hydroxylation of Polycyclic Phenols

Engineering a Feruloyl–Coenzyme A Synthase for Bioconversion of Phenylpropanoid Acids into High-Value Aromatic Aldehydes

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