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10.1002/anie.201904923
Angewandte Chemie International Edition
COMMUNICATION
A Novel Chemo-Enzymatic Cascade for Smart Detection of Nitro-
and Halogenated Phenols
Pratchaya Watthaisong[a], Pornkanok Pongpamorn[a], Panu Pimviriyakul[a], Somchart Maenpuen[b],
Yoshihiro Ohmiya[c], Pimchai Chaiyen*[a]
HP substituents can lead to acute and severe diseases such as
cancer.[4,5]
Abstract: The flavin-dependent monooxygenase, HadA, catalyzes
the dehalogenation and denitration of the toxicants, nitro- and
Dehalogenases are in the redox (EC 1.14) and hydrolase
(EC 3.8) classes of enzymes. They are powerful biocatalysts for
detoxifying toxic environmental pollutants[6] and their applications
in industries have been demonstrated.[7] In this work, we have
developed a new waste biodetoxification concept based on the
reaction of HadA monooxygenase, a dechlorinating flavin-
dependent monooxygenase, that can catalyze halide and nitro
group elimination from NPs and HPs to generate benzoquinone
(BQ).[8] The enzyme is encoded by the hadA gene located in the
had operon which is responsible for chlorophenol degradation in
Ralstonia pickettii.
halogenated phenols, to benzoquinone. The HadA reaction can be
applied in one-pot reactions towards the de novo synthesis of D-
luciferin by coupling with D-Cys condensation. D-luciferin, a valuable
chemical widely used in biomedical applications, can be used as a
substrate for the reaction of firefly luciferase to generate
bioluminescence. As nitro- and halogenated phenols are key
indicators of human overexposure to pesticides commonly used
worldwide and indicators of pesticide contamination, the technology
provides a sensitive and convenient tool for improved biomedical and
environmental detection at ppb sensitivity in biological samples
without the requirement for any pre-treatment. This newly developed
methodology provides the dual-pronged advantage of waste
biodetoxification to produce a valuable chemical as well as a smart
detection tool for environmental and biomedical detection.
It was previously reported that chemical condensation of BQ
and D-cysteine (D-Cys) can result in D-luciferin, although with a
very low yield (~ 0.3%).[9] We thus linked this enzymatic and
chemical condensation to turn toxicants into D-luciferin via one-
pot chemo-enzymatic reaction. The current price of D-luciferin is
206 USD per mg (Sigma) with a total market size of 18 billion USD
annually.[10] D-luciferin is also widely used in biomedical
research[11] and biodetection[12]; more than 2420 publications in
2018 reported the use of D-luciferin in their experiments
(Supporting Information, Figure S1). As D-luciferin formation can
be further applied to generate bioluminescence signals by firefly
luciferase (Fluc), the chemo-enzymatic cascade developed also
offers additional value in providing detection technology as an
integrative biodetoxification-biosynthesis platform for biodetection
of NP and HP, which are metabolites and biomarkers of
pesticides/herbicides (Figure 1). Bioluminescence is a powerful
detection technology because it provides high signals with low
non-specific background and is widely used as a diagnostic
tool.[13]
Urban development and use of chemicals in various industries
are major factors for chemical contamination in environment
worldwide. Biocatalysis provides powerful and sustainable technology
for conversion of toxicants to less toxic products which, in some cases,
may lead to economical advantage.[1] Halogenated and nitro-
aromatic compounds are widely used in household and industrial
settings including dyes, plasticizers, explosives, pharmaceuticals,
flame retardants, disinfectants, chemical-warfare agents,
pesticides and herbicides.[2] Spillage of nitrophenols (NPs) and
halogenated phenols (HPs) in manufacturing areas, and their
long-term accumulation in the environment as a result of pesticide
and herbicide degradation,[3] has long been recognized as
causing adverse effects in humans and wildlife. Consumption of
food contaminated with pesticides/herbicides containing NP and
[a]
P. Watthaisong, P. Pongpamorn, P. Pimviriyakul, P. Chaiyen
School of Biomolecular Science & Engineering (BSE),
Vidyasirimedhi Institute of Science and Technology (VISTEC),
Wangchan Valley,Rayong 21210, Thailand
We first established an enzymatic cascade of HadA to
convert 4-nitrophenol (4-NP, 2a), 4-fluorophenol (4-FP, 2b), 4-
chlorophenol (4-CP, 2c), 4-bromophenol (4-BrP, 2d) and 4-
iodophenol (4-IP, 2e) into BQ. As HadA requires constant
generation of reduced FAD (FADH-) in addition to other co-
substrates, molecular oxygen and NP or HP (Figure 2 A), two
[b]
[c]
S. Maenpuen
Department of Biochemistry, Faculty of Science,
Burapha University, Chonburi 20131, Thailand
Y. Ohmiya
National Institute of Advanced Industrial Science
and Technology (AIST), Tsukuba, Ibaraki 305-8566, Japan.
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