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

Abstract

The hydroxylation of phenols into polyphenols, which are valuable chemicals and pharmaceutical products, is a challenging reaction. The search for green synthetic processes has led to considering microorganisms and pure hydroxylases as catalysts for phenol hydroxylation. Herein, we report the structural and functional characterization of the flavin adenine dinucleotide (FAD)‐dependent 4‐hydroxyphenylacetate 3‐monooxygenase from Escherichia coli, named HpaB. It is shown that this enzyme enjoys a relatively broad substrate specificity, which allows the conversion of a number of non‐natural phenolic compounds, such as tyrosol, hydroxymandelic acid, coumaric acid, hydroxybenzoic acid and its methyl ester, and phenol, into the corresponding catechols. The reaction can be performed by using a simple chemical assay based on formate as the electron donor and the organometallic complex [Rh(bpy)Cp*(H2O)]2+ (Cp*: 1,2,3,4,5‐pentamethylcyclopentadiene, bpy: 2,2′‐bipyridyl) as the catalyst for FAD reduction. The availability of a crystal structure of HpaB in complex with FAD at 1.8 Å resolution opens up the possibility of the rational tuning of the substrate specificity and activity of this interesting class of phenol hydroxylases.

A Heterogeneous Recyclable Rhodium‐based Catalyst for the Reduction of Pyridine Dinucleotides and Flavins

“…We recently reported an enzymatic aromatic hydroxylation system converting a variety of phenols into catechols, based on the two‐component flavin‐dependent 4‐hydroxyphenylacetate monooxygenase from Escherichia coli . This system consists of two enzymes: (i) a flavin reductase, named Fre, which catalyzes the reduction of FAD into FADH 2 by NADH; (ii) an hydroxylase, named HpaB, which uses O 2 to convert the reduced flavin FADH 2 into the corresponding flavin hydroperoxide for subsequent hydroxylation of hydroxyphenylacetate (HPA) into dihydroxyphenylacetate (DHPA) (Scheme ).…”

“…For comparison with a homogeneous system we used the previously reported [Cp*Rh(bpy)(H 2 O)] 2+ /formate system and the results are shown in Figure S7. Using a concentration of 100 μM of the soluble Rh complex led to reduction of 500 μM NAD + with the highest initial rate of about 8 μM ⋅ min −1 .…”

“…NADH, NADPH, NAD + , NADP + , FAD, FMN and Riboflavin were purchased from Sigma‐Aldrich, Bpy‐PMO from TCI Europe N.V., [Cp*RhCl 2 ] 2 from Sigma‐Aldrich. HpaB from Escherichia coli was purified according to a recently reported procedure …”

“…We recently reported an enzymatic aromatic hydroxylation system converting a variety of phenols into catechols, based on the two-component flavin-dependent 4-hydroxyphenylacetate monooxygenase from Escherichia coli. [8] This system consists of two enzymes: (i) a flavin reductase, named Fre, which catalyzes the reduction of FAD into FADH 2 by NADH; (ii) an hydroxylase, named HpaB, which uses O 2 to convert the reduced flavin FADH 2 into the corresponding flavin hydroperoxide for subsequent hydroxylation of hydroxyphenylacetate (HPA) into dihydroxyphenylacetate (DHPA) (Scheme 2). Because the system consumes stoichiometric amounts of NADH, an expensive reagent, we developed a simple assay in which FADH 2 was continuously regenerated though reduction of FAD by formate catalyzed by the homogeneous organometallic complex {[Cp*Rh(bpy)(H 2 O)] 2 + } complex (OC), thus excluding NADH and Fre.…”

“…In the absence of such a regeneration system, more expensive and complex scenarios have to be set up: the reduced flavin, an expensive and unstable compound, is either used stoichiometrically or produced at the expense of stoichiometric amounts of NAD(P)H, thanks to the addition of another enzyme, a NAD(P)H:flavin oxidoreductase (also named flavin reductase). Nice examples of the utilization of the OC/FA system for fueling such a two‐component monooxygenase system, composed of a flavin reductase component and a hydroxylase component, have been reported in the case of epoxidation and aromatic hydroxylation, more recently by us, reactions.…”