Altering 2‐Hydroxybiphenyl 3‐Monooxygenase Regioselectivity by Protein Engineering for the Production of a New Antioxidant

Abstract

2-Hydroxybiphenyl 3-monooxygenase is a flavin-containing NADH-dependent aromatic hydroxylase that oxidizes a broad range of 2-substituted phenols. In order to modulate its activity and selectivity, several residues in the active site pocket were investigated by saturation mutagenesis. Variant M321A demonstrated altered regioselectivity by oxidizing 3-hydroxybiphenyl for the first time, thus enabling the production of a new antioxidant, 3,4-dihydroxybiphenyl, with similar ferric reducing capacity to the well-studied piceatannol. The crystal structure of M321A was determined (2.78 Å), and molecular docking of the 3-substituted phenol provided a rational explanation for the altered regioselectivity. Furthermore, HbpA was found to possess pro-S enantioselectivity towards the production of several chiral sulfoxides, whereas variant M321F exhibited improved enantioselectivity. Based on the biochemical characterization of several mutants, it was suggested that Trp97 stabilized the substrate in the active site, Met223 was involved in NADH entrance or binding to the active site, and Pro320 might facilitate FAD movement.

Non-Native Site-Selective Enzyme Catalysis

“…266 A more recent study showed that HbpA M321A catalyzes selective C-4 hydroxylation of 3-hydroxybiphenyl (240), a substrate not accepted by wild-type HbpA (Scheme 42A). 267 The 4-hydroxyphenylacetate 3-hydroxylases (HPAHs) are two component FMOs that natively catalyze hydroxylation of 4-hydroxyphenylacetate to 3,4-dihydroxyphenylacetate and accept a range of additional substrates. 268 The S146A variant of HPAH from Acinetobacter baumannii provides improved activity on 4-aminophenylacetic acid to give 3-hydroxy-4-aminophenylacetic acid when compared to wild-type.…”

Oxygenating Biocatalysts for Hydroxyl Functionalisation in Drug Discovery and Development

“…Fishman et al. produced the HbpA variant, M321A, through saturation mutagenesis studies, which displayed altered regioselectivity and activity [89] . M321A catalysed hydroxylation of non‐natural substrate 3‐hydroxybiphenyl ( 7 ) to produce a new antioxidant, 3,4‐hydroxybiphenyl ( 8 ) (Figure 3A) [89] …”

“…[88] Fishman et al produced the HbpA variant, M321A, through saturation mutagenesis studies, which displayed altered regioselectivity and activity. [89] M321A catalysed hydroxylation of non-natural substrate 3-hydroxybiphenyl (7) to produce a new antioxidant, 3,4-hydroxybiphenyl (8) (Figure 3A). [89] Unlike single-component monooxygenases, which have only been reported to use FAD as a cofactor, two-component monooxygenases can either use reduced FAD or FMN.…”

“…[89] M321A catalysed hydroxylation of non-natural substrate 3-hydroxybiphenyl (7) to produce a new antioxidant, 3,4-hydroxybiphenyl (8) (Figure 3A). [89] Unlike single-component monooxygenases, which have only been reported to use FAD as a cofactor, two-component monooxygenases can either use reduced FAD or FMN. [80] 4hydroxyphenylacetate 3-hydroxylases (HPAHs) are extensively-studied two-component monooxygenases which catalyse the hydroxylation of 4-hydroxyphenylacetate (4-HPA) to yield 3,4dihydroxyphenylacetate (3,4-DHPA).…”

Flavoprotein Monooxygenases and Halogenases

“…3-hydroxybenzoate 4-hydroxylase (3HB4H; EC 1.14.13.23) and 3-hydroxybenzoate 6-hydroxylase (3HB6H; EC 1.14.13.24). The aromatic hydroxylase 2-hydroxybiphenyl 3-monooxygenase (HbpA; EC 1.14.13.44) has been studied extensively for applied biocatalysis (Scheme 7.2a) [36][37][38]. An important hydroxylation reaction catalyzed by several group A enzymes concerns the ipso-attack [46].…”