p-Hydroxyphenylacetate 3-Hydroxylase as a Biocatalyst for the Synthesis of Trihydroxyphenolic Acids

Abstract

Trihydroxyphenolic acids such as 3,4,5-trihydroxycinnamic acid (3,4,5-THCA) 4c and 2-(3,4,5-trihydroxyphenyl)acetic acid (3,4,5-THPA) 2c are strong antioxidants that are potentially useful as medicinal agents. Our results show that p-hydroxyphenylacetate (HPA) 3-hydroxylase (HPAH) from Acinetobacter baumannii can catalyze the syntheses of 3,4,5-THPA 2c and 3,4,5-THCA 4c from 4-HPA 2a and p-coumaric acid 4a, respectively. The wildtype HPAH can convert 4-HPA 2a completely into 3,4,5-THPA 2c within 100 min (total turnover number (TTN) of 100). However, the wild-type enzyme cannot efficiently synthesize 3,4,5-THCA 4c. To improve the efficiency, the oxygenase component of HPAH (C 2 ) was rationally engineered in order to maximize the conversion of p-coumaric acid 4a to 3,4,5-THCA 4c. Results from site-directed mutagenesis studies showed that Y398S is significantly more effective than the wildtype enzyme for the synthesis of 3,4,5-THCA 4c; it can catalyze the complete bioconversion of p-coumaric acid 4a to 3,4,5-THCA 4c within 180 min (TTN ∼ 23 at 180 min). The yield and stability of 3,4,5-THPA 2c and 3,4,5-THCA 4c were significantly improved in the presence of ascorbic acid. Thermostability studies showed that the wild-type C 2 was very stable and remained active after incubation at 30, 35, and 40°C for 24 h. Y398S was moderately stable because its activity was retained for 24 h at 30°C and for 15 h at 35°C. Transient kinetic studies using stopped-flow spectrophotometry indicated that the key improvement in the reaction of Y398S with p-coumaric acid 4a lies within the protein−ligand interaction. Y398S binds to pcoumaric acid 4a with higher affinity than the wild-type enzyme, resulting in a shift in equilibrium toward favoring the productive coupling path instead of the path leading to wasteful flavin oxidation.

Structural and Functional Characterization of 4‐Hydroxyphenylacetate 3‐Hydroxylase from Escherichia coli

“…However, the comparison is limited to a very small number of the same substrates as those tested here and the other studies, namely, HPA, Ph, Cou, and HPG . It is also possible to compare the substrate specificity properties of EcHpaB with those of C 2 ‐HpaH from A. baumanii because the latter has also been studied in a pure enzyme system . It appears that C 2 ‐HpaH accepts much fewer substrates than that of EcHpaB: among the few phenols that can be hydroxylated, apart from HPA, HBA and Cou are much less efficiently converted into the corresponding catechols, compared with EcHpaB.…”

Kinetic Mechanism of the Dechlorinating Flavin-dependent Monooxygenase HadA

“…Furthermore, the individual rate constants associated with each step and the mechanism of reduced flavin transfer between the two components have been reported (24 -30). This knowledge has been useful for applying HPAH for the synthesis of bioactive phenolic acids and in the hydroxylation of aniline derivatives (31,32).…”

Biotransformation of Plant-Derived Phenolic Acids

“…Hydroxylation of plant-derived PAs such as pCA results directly in production of CA. [94] The same enzyme can also hydroxylate other PAs such as 3-(4-hydroxyphenyl)propionic acid to form 3-(3,4dihydroxyphenyl)propionic acid and 3-(3,4,5-trihydroxyphenyl) propionic acid, p-hydroxyphenylacetic acid to form 3,4-dihydroxyphenylacetic acid and 3,4,5-trihydroxyphenylacetic acid, and pHBA to form 3,4-dihydroxybenzoic acid and 3,4,5trihydroxybenzoic acid or GA (Figure 2). [87,88] Whole-cell biocatalysis using E. coli expressing the F185L mutant of Cytochrome P450 CYP199A2 can produce CA with a yield of 2.8 g L À1 , [89] whole-cell biocatalysis using E. coli expressing the hpaBC gene encoding a two-component flavindependent monooxygenase from Pseudomonas aeruginosa is even more effective, yielding up to 10.2 g L À1 of CA within 24 h. [90] 3,4,5-Trihydroxycinnamic acid is a strong antioxidant, having greater antioxidant activity in aqueous solution than its monoand dihydroxyphenolic acid counterparts.…”

“…Native microorganisms including Pycnoporus cinnabarinus and Streptomyces caeruleus have been used to synthesize CA from pCA with production yields of 21% (257 mg L À1 ) and 16.6% (150 mg L À1 ), respectively. [94,95] As the wild-type enzyme cannot use pCA efficiently as a substrate, rational engineering was carried out to obtain the Y398S variant that is more efficient than the wild-type enzyme in producing 3,4,5-trihydroxycinnamic acid from pCA with a yield of 23 mg L À1 . [91] Therefore, the compound has great potential for use as an antioxidant in the food and pharmaceutical industries.…”

“…[87,88] Whole-cell biocatalysis using E. coli expressing the F185L mutant of Cytochrome P450 CYP199A2 can produce CA with a yield of 2.8 g L À1 , [89] whole-cell biocatalysis using E. coli expressing the hpaBC gene encoding a two-component flavindependent monooxygenase from Pseudomonas aeruginosa is even more effective, yielding up to 10.2 g L À1 of CA within 24 h. [90] 3,4,5-Trihydroxycinnamic acid is a strong antioxidant, having greater antioxidant activity in aqueous solution than its monoand dihydroxyphenolic acid counterparts. [94] Recently, an efficient process to extract pCA and CA from palm oil mill effluent, a renewable and sustainable feedstock, was developed and followed by their conversion to 3,4,5-trihydroxycinnamic acid. Recently, it has been shown that the flavin-dependent p-hydroxyphenylacetate 3-hydroxylase from Acinetobacter baumannii [92,93] can be used as a biocatalyst for the synthesis of CA and 3,4,5-trihydroxycinnamic acid from pCA in vitro.…”