4-Hydroxyphenylacetate 3-hydroxylase (HpaB) has high potential for use in polyphenol synthesis via ortho-hydroxylation. Although the HpaB enzymes from Pseudomonas aeruginosa (PaHpaB) and Escherichia coli (EcHpaB) have been well studied, few studies have compared their activity and substrate selectivity. Thus, which HpaB is optimal for use in the biotechnological production of polyphenols is unclear. In this study, we performed a comparative analysis of the substrate- and regio-selectivity of PaHpaB, EcHpaB, and the recently discovered enzyme from Rhodococcus opacus (RoHpaB). The activity of these enzymes was first compared toward representative aromatic substrates. PaHpaB and EcHpaB exhibited very similar catalytic activity toward p-coumaric acid and tyrosol with one benzene ring, whereas PaHpaB exhibited greater activity than EcHpaB toward resveratrol and naringenin with two benzene rings. These results suggest that PaHpaB is superior to EcHpaB in converting bulky compounds. Furthermore, PaHpaB also exhibited catalytic activity toward a flavonoid, daidzein (7,4'-dihydroxyisoflavone), whereas EcHpaB did not. RoHpaB also exhibited strong activity toward daidzein in addition to other aromatic substrates. Interestingly, PaHpaB hydroxylated the 6-position of daidzein, whereas RoHpaB hydroxylated the 3'-position. PaHpaB and RoHpaB enabled the facile synthesis of not only 6-hydroxydaidzein and 3'-hydroxydaidzein but also 6,3'-dihydroxydaidzein via the cascade reaction. This study is the first to demonstrate synthesis of hydroxydaidzeins using HpaB enzymes.
Keywords: Caffeic acid; HpaBC; Hydroxydaidzein; Monooxygenase; Piceatannol.
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