A hydroquinone-specific screening system for directed P450 evolution

Abstract

The direct hydroxylation of benzene to hydroquinone (HQ) under mild reaction conditions is a challenging task for chemical catalysts. Cytochrome P450 (CYP) monooxygenases are known to catalyze the oxidation of a variety of aromatic compounds with atmospheric dioxygen. Protein engineering campaigns led to the identification of novel P450 variants, which yielded improvements in respect to activity, specificity, and stability. An effective screening strategy is crucial for the identification of improved enzymes with desired characteristics in large mutant libraries. Here, we report a first screening system designed for screening of P450 variants capable to produce hydroquinones. The hydroquinone quantification assay is based on the interaction of 4-nitrophenylacetonitrile (NpCN) with hydroquinones under alkaline conditions. In the 96-well plate format, a low detection limit (5 μM) and a broad linear detection range (5 to 250 μM) were obtained. The NpCN assay can be used for the quantification of dihydroxylated aromatic compounds such as hydroquinones, catechols, and benzoquinones. We chose the hydroxylation of pseudocumene by P450 BM3 as a target reaction and screened for improved trimethylhydroquinone (TMHQ) formation. The new P450 BM3 variant AW2 (R47Q, Y51F, I401M, A330P) was identified by screening a saturation mutagenesis library of amino acid position A330 with the NpCN assay. In summary, a 70-fold improved TMHQ formation was achieved with P450 BM3 AW2 when compared to the wild type (WT) and a 1.8-fold improved TMHQ formation compared to the recently reported P450 BM3 M3 (R47S, Y51W, A330F, I401M).Electronic supplementary materialThe online version of this article (10.1007/s00253-018-9328-3) contains supplementary material, which is available to authorized users.

Directed Evolution of P450 BM3 towards Functionalization of Aromatic O-Heterocycles

“…The P450 BM3 engineering strategy is summarized in Figure 3, where screening of previously prepared in-house epPCR and site-saturation-mutagenesis (SSM) libraries of P450 BM3 WT [44,47,53] yielded P450 BM3 CM1 (R255P/P329H), which was subjected to two rounds of epPCR to identify additional beneficial positions. This led to the identification of four positions in total (I122, R255, P329, and F331) that were selected for individual site saturation mutagenesis (SSM) using WT as template (Figure A5).…”

“…However, such protein engineering campaigns usually generate thousands of variants, where a major challenge is the development of product-based screening systems to reliably identify better performing catalysts, i.e., the screening system has to be of high throughput, reproducible, and optimized for sensitivity of the desired function. Traditionally, enzyme activity is determined in 96-microtiter plates (MTPs) using either crude cell lysates or purified enzyme to perform product-based colorimetric or fluorometric assays (e.g., 4-aminoantipyrine for phenolic compound detection [43], NpCN for the detection of specific hydroquinones [44], pNTP for styrene epoxidation [45], or fluorescence for the detection of steroid hydroxylation [46]). A generally applicable and emerging possibility is 96 multiplex-capillary electrophoresis (CE), which has been added to the range of suitable screening systems for P450-directed evolution campaigns [47].…”

A 96-multiplex capillary electrophoresis screening platform for product based evolution of P450 BM3

“…To ensure P450 BM3 product formation above the lower detection limit, NADPH regeneration was initiated by supply of glucose dehydrogenase (4 U mL −1 ) and glucose (60 mM) similar as published by Weingartner et al . 11 . Different NADPH regeneration times (3–18 hours) were investigated and 18 hours was identified as most suitable incubation interval for subsequent screening towards 4-hydroxy-isophorone formation.…”

“…Product based colorimetric and fluorescent screening systems have been developed in case of P450s for the detection of hydroquinones 11 , epoxides 12 , phenols 13,14 and indigo 15 as well as for enantioselectivity 16 . Alternatives with low throughput are analytical methods like high performance liquid chromatography (HPLC), gas chromatography (GC) or mass spectrometry (MS) 17–19 .…”

A Promiscuous Bacterial P450: The Unparalleled Diversity of BM3 in Pharmaceutical Metabolism

“…WT BM3 and the selected variants also selectively hydroxylate the methyl-substituted benzene derivatives, pseudocumene and mesitylene, to the phenolic building blocks used in the α-tocopherol synthesis via quinolone intermediates. For example, the introduction of the point mutation A330F increased the selectivity towards metabolite trimethylhydroquinolone to 75% [ 87 , 88 ]. The selective hydroxylation of various halogenated alkyl benzene derivatives by R47L/Y51F/A330P yields key intermediates such as 2-chlorotoluene and 4-methyl-2-ethylphenol with turnovers of 500 to 1600 min −1 .…”