Enantioselective Catabolism of Napropamide Chiral Enantiomers in Sphingobium sp. A1 and B2

Article abstract of DOI:10.1021/acs.jafc.9b00707

Napropamide [N,N-diethyl-2-(1-naphthalenyloxy)propenamide, NAP] is a highly efficient and broad-spectrum amide herbicide. Little is known about the bacterial catabolism of its different enantiomers. Here, we report the isolation of two NAP-degrading strains of Sphingobium sp., A1 and B2, and the different catabolic pathways of different enantiomers in these two strains. Strain A1 dioxygenated NAP at different positions of the naphthalene ring of different enantiomers, leading to the complete degradation of R-NAP while producing a dead-end product from S-NAP. Strain B2 cleaved the amido bonds of both enantiomers, but only the product from S-NAP could be further transformed to form α-naphthol and mineralize in strain B2. The degradation rates of R-NAP and S-NAP in the combination degradation by strains A1 and B2 were 24.8 and 7.5 times that in the single-strain degradation by strain B2 or A1, respectively, showing enhanced synergistic catabolism between strains A1 and B2. This study provides new insights into the enantioselective catabolic network of the chiral herbicide NAP in microorganisms.

Full text of DOI:10.1021/acs.jafc.9b00707

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Biotechnology and Biological Transformations
Enantioselective catabolism of napropamide
chiral enantiomers in Sphingobium sp. A1 and B2
Jun-Wei Huang, Dian Chen, Xiao-Kun Cheng, Gui-Ping Liu, Guo-xiang Wang, and Jian-Dong Jiang
J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.9b00707 • Publication Date (Web): 28 May 2019
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Enantioselective catabolism of napropamide chiral enantiomers in Sphingobium
sp. A1 and B2
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Junwei Huanga, Dian Chena, Xiaokun Chenga, Guiping Liua, Guoxiang Wangc,
Jiandong Jianga,b,*
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aDepartment of Microbiology, Key Lab of Microbiology for Agricultural Environment,
Ministry of Agriculture, College of Life Sciences, Nanjing Agricultural University,
210095, Nanjing, China.
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bJiangsu Key Lab for Solid Organic Waste Utilization, 210095, Nanjing, China.
cLaboratory Centre of Life Science, College of Life Sciences, Nanjing Agricultural
University, 210095, Nanjing, China.
Address correspondence to Jian-Dong Jiang, jiang_jjd@njau.edu.cn;
Tel: +86-025-84399726; Fax: +86-025-84396314
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Abstract
Napropamide (N,N-diethyl-2-(1-naphthalenyloxy)propenamide, NAP) is a highly
efficient and broad-spectrum amide herbicide. Little is known about the bacterial
catabolism of its different enantiomers. Here, we report the isolation of two NAP-
degrading strains of Sphingobium sp., A1 and B2, and the different catabolic pathways
of different enantiomers in these two strains. Strain A1 di-oxygenated NAP at different
positions of the naphthalene ring of different enantiomers, leading to the complete
degradation of R-NAP, while producing a dead-end product from S-NAP. Strain B2
cleaved the amido bonds of both enantiomers, but only the product from S-NAP could
be further transformed to form α-naphthol and mineralize in strain B2. The degradation
rates of R-NAP and S-NAP in the combination degradation by strain A1 and B2 were
24.8- and 7.5-times that in the single strain degradation by strain B2 or strain A1,
respectively, showing enhanced synergistic catabolism between strains A1 and B2. This
study provides new insights into the enantioselective catabolic network of the chiral
herbicide NAP in microorganisms.
Key words: Synergistic catabolism; Stereospecific; Napropamide; Mineralized
consortium
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Introduction
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Chiral pesticides comprise 28% of the global pesticide market. In China, the proportion
of chiral pesticides has increased from 19% during the 1980s to approximately 40%
currently.1, 2 There are 14 classes and 19 major types of herbicides with chiral centers,
accounting for a greater proportion in pesticides. Because of the enantiomeric
selectivity from the different effects of the enantiomers on target and non-target
organisms, different chiral isomers have been reported to show different biological
activities, environmental toxicities, and environmental behaviors.3, 4 For example, the
demethylation of (S)-dichlorprop methyl in soil is faster than that of (R)-dichlorprop
methyl, leading to plant phytotoxicity because of the accumulation of the highly
herbicidal (R)-dichlorprop methyl in the soil.3 Some chiral herbicides show the opposite
physiological effects.4 For instance, the R-enantiomer of the herbicide diclofop is
effective in weed control, while the S-enantiomer has no weeding effects but shows
more toxicity toward algae than the R-enantiomer.5, 6 The microbial community is one
of the important factors affecting the biodegradation of chiral herbicides. Under
nonsterilized conditions, more than 99% of the (R)- and (S)-indoxacarb are dissipated
after 75 days of incubation in acidic soil. However, only 5-10% of the initial
concentration of (R)- and (S)-indoxacarb are degraded after 75 days of incubation in
sterilized acidic soil.7 Moreover, R- and S-enantiomers can be inter-converted by soil
microorganisms.8, 9 (S)-indoxacarb had a significantly higher inversion rate to (R)-
indoxacarb than its antipode in alkaline soil.7 Because most of the chiral pesticides are
chemically synthesized in racemate for use, they inevitably release all isomers into the
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environment, despite their activity and toxicity. Consequently, there is an increasing
concern of the pollution risk of different isomers of chiral pesticides in the
environment.10 The environmental fate of different isomers, especially their catabolism
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by microorganisms, needs extensive investigation.
Napropamide (N,N-diethyl-2-(naphthalen-1-yloxy)propanamide) is a selective
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systemic amide herbicide used in soils to control several annual grasses and broad-
leaved weeds.11 Napropamide (NAP) is characterized by its efficiency and broad
spectrum and selectivity,11, 12 and it is also found in formulations with other pesticides
such as monolinuron, nitralin, simazine, trifluralin, tefluthrin, and tebutam. NAP is a
typical chiral herbicide and contains two enantiomers, R-NAP and S-NAP (Figure S1).
Because the chemically synthesized racemic NAP (Rac-NAP) is commonly used, both
enantiomers enter the environment through point and non-point sources and are
distributed in water, soil, sediment and biota.13 The NAP concentrations detected in the
environment range from 0.1 to 0.46 mg/kg.14, 15 A recent study showed that R-NAP had
the strongest herbicidal activity, followed by Rac-NAP, while S-NAP was the weakest.
The inhibition effect of R-NAP on the root growth of Echinochloa crusgalli L. was 9.4
times higher than that of S-NAP at the concentration of 0.05 mg/L.16 Meanwhile, the
toxicity of S-NAP toward Microcystis aeruginosa (EC20<0.1 mg/L) was notably higher
than that of R-NAP (EC20= 0.1-1.0 mg/L).16 The molecular docking of NAP
enantiomers with the thyroxine receptor (TR) showed that S-NAP combined more
closely with TR, leading to higher toxicity toward organisms.16 Therefore, S-NAP tends
to cause a much more serious threat to ecological systems than R-NAP, although both
of them are negative to ecosystems. Although microbial degradation has been
recognized as the key pathway for the dissipation of NAP from the environment,17 NAP
catabolism via microorganisms has not been extensively studied. Bacillus sp. LGY06
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is the only pure strain reported to be capable of degrading Rac-NAP. However, the
complete catabolic pathway of Rac-NAP in strain LGY06 has not been elucidated.18
Moreover, the enantioselective catabolism of the two enantiomers of NAP has never
been studied.18
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In this study, the different catabolic efficiencies and pathways of the two enantiomers
of Rac-NAP in the two strains of Sphingobium sp. A1 and B2 isolated from a Rac-NAP-
contaminated soil were systematically studied. The enantioselective catabolism of
different NAP chiral isomers in strains A1 and B2 was found, and these strains could
use complementary pathways to enhance the degradation of Rac-NAP and synergistic
catabolism to accelerate the degradation of Rac-NAP. Our study provides new insights
into the enantioselective catabolic network of chiral NAP in microorganisms.
Materials and Methods
1. Chemical reagents, primers, and media
Rac-NAP (99.7%) was purchased from Shanghai Pesticide Research Institute Co.,
Ltd. (Shanghai, China). Methanol (chromatography grade) and acetic acid
(chromatography grade) were purchased from EMD Millipore corporation (Darmstadt,
Germany). The other reagents used in this study were commercially available. Luria-
Bertani (LB) medium or minimal salt medium (MSM) was used to culture isolated
strains at 30°C. Streptomycin (Sm) was added to the medium at 100 mg/L to culture
Sphingobium species because of their natural resistance to streptomycin.19
2. Isolation and characterization of two bacterial strains from a Rac-NAP-contaminated
soil
Rac-NAP-contaminated soil was collected from Jiangsu Kuaida Agrochemical Co.,
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Ltd, China. The isolation of NAP-degrading strains was performed using the traditional
enrichment method as previously described with minor modifications.20 Rac-NAP (0.2
mM) was provided as the sole carbon source in MSM for enrichment. Colonies selected
from the enrichment were checked for their capacities to degrade Rac-NAP via a UV-
Visible Spectrophotometer (UV-2450, Shimadzu, Japan) and high-performance liquid
chromatography (HPLC, UltiMate 3000 RSLC, Thermo Fisher Scientific, America)
with a C18 reversed-phase column (4.6 × 250 mm, 5 μm). The separation conditions of
the HPLC were as follows: the mobile phase was a mixed solution of methanol: water:
acetic acid (75:24:1, v:v:v), the flow rate was 0.8 mL/min, the column temperature was
30°C, the detection wavelength was 250 nm, and the injection volume was 20 μL.21 All
experiments were performed in triplicate. Two strains termed A1 and B2, capable of
degrading R-NAP and/or S-NAP, were finally isolated and purified. The isolates were
identified based on their morphological, physiological and biochemical properties as
well as 16S rRNA gene analysis. The primers 27F (5’-agagtttgatcmtggctcag-3’) and
1492R (5’-tacggytaccttgttacgactt-3’) were used to amplify the nearly complete 16S
rRNA gene. The sequenced 16S rRNA gene sequences of strains A1 and B2 were
deposited in the GenBank database under the accession numbers MK411213 and
MK447612, respectively.
3. Chiral separation and identification
The enantiomers of Rac-NAP were separated using the Shimadzu LC-20AT HPLC
system (Shimadzu, Japan). The CHIRALPAK IC (Daicel Corp., Japan) was used as the
separation column (4.6 × 150 mm, 5 μm), and the mobile phase was hexane:
isopropanol (80:20, v:v) at a flow rate of 1.0 mL/min. The detection wavelength and
temperature were 231 nm and 35°C, respectively. The electronic circular dichroism
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(ECD) spectra of the NAP enantiomers were recorded using a circular dichroism
spectropolarimeter J-810 (JASCO Company, Japan) with methanol as the solvent under
a wavelength range of from 200 to 400 nm.22 The ECD plots of R-NAP and S-NAP
were simulated using SpecDis software.23 The average map was obtained by the
Boltzmann-weighted average method and then was compared with the experimental
ECD spectrum to determine the absolute configuration.24-26
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4. Synergistic catabolism via the combination of strains A1 and B2
Strains A1 and B2 were separately cultured in LB medium at 30°C until the mid-log
phase. The cells were pelleted by centrifugation at 8000 × g for 10 min and then were
washed twice with sterilized H2O. Strain A1, strain B2 or the combination of strains A1
and B2 (cell number 1:1) were inoculated to MSM with 0.2 mM Rac-NAP (0.1 mM R-
NAP plus 0.1 mM S-NAP), 0.2 mM R-NAP or 0.2 mM S-NAP, respectively, at a final
concentration of OD600 = 0.1. All treatments were incubated at 30°C and 160 rpm.
Samples (500 μL) were collected from each treatment at regular intervals and then were
mixed with an equal volume of methanol and completely vortexed for 2 min. After
centrifugation at 15,000 × g for 3 min, the concentration of each added substrate (Rac-
NAP, R-NAP, and S-NAP) in the supernatant was determined using HPLC analysis.
Cell growth was determined via OD600 measurement. Inoculation with sterilized strain
A1 or B2 cells under the same conditions was used as the negative control. All the
experiments were performed in triplicate.
5. Metabolic analysis and identification
Strain A1 or strain B2 was incubated in MSM containing 0.2 mM R-NAP or S-NAP
at an initial OD600 = 0.1. A sample (1 mL) of the S-NAP degradation by strain A1 was
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collected at 48 h, while other samples (1 mL) were collected at 6 h. Each collected
sample was mixed with an equal methanol volume. After filtration using a 0.22-μm
membrane (Millipore, USA), the metabolites were identified using HPLC and liquid
chromatography-tandem mass spectrometry (LC-MS/MS). For the HPLC-MS/MS
analysis, the HPLC system (Shimadzu, Japan) was connected to a TripleTOF 5600 mass
spectrometer (AB SCIEX, American) that was equipped with an electrospray ionization
(ESI) probe. The HPLC column was a Kinetex C18 (100 mm × 2.1 mm, particle size 2.6
μm). The mobile phase consisted of solvents A (ultrapure water) and B (methanol) with
a gradient program that started by maintaining 30% B for 3 min, followed by increasing
to 75% B from 3 to 15 min, maintaining 75% B from 15 to 30 min, and then returning
to 30% B within 5 min. The flow rate was 0.2 mL/min. The injection volume was 10
μL, and the compounds were first ionized in negative or positive polarity based on their
features.
To collect and purify compound 1, strain A1 was cultured and harvested according
to the method of Chen et al.27 The cells were inoculated into MSM medium
supplemented with 0.2 mM S-napropamide with an initial OD600 of 2.0. The cultures
were shaken in an incubator at 180 rpm and 30°C. The crude extract of compound 1
was collected as described previously when the substrate was completely degraded.28
The crude extract of compound 1 was purified using a silica gel column using petroleum
ether:acetone (35:1 to 10:1, v/v) as an eluent, followed by elution with acetone to yield
compound 1. 1H nuclear magnetic resonance (NMR) (δ), 1H-1H correlation
spectroscopy (COSY), heteronuclear single quantum coherence (HSQC), heteronuclear
multiple bond correlation (HMBC), and nuclear Overhauser effect spectroscopy
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(NOESY) data (δ) were measured in Acetone-d6 at 400 MHz, and C NMR data (δ)
were measured in Acetone-d6 at 100 MHz using Bruker Avance spectrometers (Bruker
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BioSpin GmbH, Beijing, China).
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6. Cell-free extract preparation and amidohydrolase activity assay in strain B2
Strain B2 cells were grown in 100 mL of LB medium containing 100 mg/L of Sm for
12 h. After centrifugation at 8000 × g for 10 min, the cells were washed twice with
deionized water and resuspended in 15 mL of 50 mM phosphate buffer
(Na2HPO4/NaH2PO4, pH 7.4) and disrupted via sonication (Ultrasonic Cell Crusher,
Thermo Fisher Scientific, USA). The disrupted cell suspension was centrifuged at
14,000 × g at 4°C for 30 min, and the supernatant was used as the cell-free extract for
the amidohydrolase activity assay. A denatured crude enzyme via boiling was prepared
as the negative control. The standard enzymatic reaction was performed as previously
described with minor modifications.27 The reaction was stopped by boiling for 1 min.
The amidohydrolase activity toward the two enantiomers of NAP was determined using
HPLC analysis. One unit of activity (U) was defined as the amount of enzyme that
catalyzed the hydrolysis of 1 μmol of R-NAP or S-NAP per minute. Specific activity
was expressed as units per milligram of cell-free extract. The kinetic parameters Km and
Vmax were calculated using the substrates within a concentration range of 0.1 -1 mM.
All the experiments were performed in triplicate.
Results
1. Isolation and identification of two NAP-degrading strains A1 and B2
When the enrichment was subcultured for three rounds, the 0.2 mM Rac-NAP in the
enrichment nearly disappeared as detected by UV-Visible spectroscopy (Figure 1A).
The enrichment was diluted and plated onto MSM agar containing 1 mM Rac-NAP at
30°C. Only four morphologically different colonies appeared on the plates after 5 days
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of incubation. These four distinct colonies were selected and checked for their
capacities to degrade NAP; only two strains, designated A1 and B2, were found to be
capable of degrading NAP. Strains A1 and B2 were both strictly aerobic, Gram-negative
and rod-shaped. Strain A1 was approximately 1.3-1.6 μm in length and 0.6-0.7 μm in
width, and strain B2 was approximately 1.3-1.7 μm in length and 0.6-0.7 μm in width.
Strain A1 was determined to be related to the Sphingobium species lineage and was
closely clustered with Sphingobium hydrophobicum C1T and Sphingobium
xenophagum NBRC 107872T, with 16S rRNA gene identities of 99.65% and 99.57%,
respectively. The 16S rRNA gene sequence of strain B2 shared 99.93% and 99.86%
identities with those of Sphingobium xenophagum NBRC 107872T and Sphingobium
hydrophobicum C1T, respectively. Based on the above phenotypic characteristics and
phylogenetic analyses, strains A1 and B2 were finally identified as two different species
of the genus Sphingobium. Interestingly, neither of the two isolates could mineralize
Rac-NAP, while the combination of strains A1 and B2 enhanced degradation of 0.2 mM
Rac-NAP (Figure 1B).
2. Chiral separation of the two enantiomers of NAP
Rac-NAP was separated by the CHIRALPAK IC (IC00CD-NA012) column of the
Shimadzu LC-20AT HPLC system, and two peaks with a retention time of 4.8 min
(peak 1) and 6.7 min (peak 2) appeared in the chromatogram (Figure S3). The
compounds corresponding to peaks 1 and 2 were collected, and the solvent was
removed using rotary evaporation to obtain the pure enantiomers (ee > 99%). The
experimental ECD spectra of peak 1 was consistent with the calculated ECD spectra of
R-NAP and experimental ECD spectra of peak 2 was consistent with the calculated
ECD spectra of S-NAP. Based on these results, the two compounds corresponding to
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peaks 1 and 2 respectively were identified as R-NAP and S-NAP. (Figure 2).
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3. Catabolism of NAP via strains A1 and B2
Strain A1 could use Rac-NAP and R-NAP as the sole carbon source for growth in
MSM (Figure S4A), while strain B2 could not use R/S/Rac-NAP for growth (Figure
S4B). Strain A1 completely degraded 0.2 mM R-NAP and S-NAP over 12 and 108 h,
respectively, showing a degrading preference to R-NAP (Figure 3A). In contrast to
strain A1, strain B2 completely degraded 0.2 mM S-NAP over 7 h, while no obvious
degradation of R-NAP was observed during this time (Figure 3B). Strain B2 showed a
weak R-NAP degradation ability, and it degraded 49.8% of the 0.2 mM R-NAP over
156 h when the cells were inoculated at OD600 =2.0 (Figure 3D). The combination of
strains A1 and B2 completely degraded 0.2 mM Rac-NAP, R-NAP or S-NAP over 6, 12
and 12 h (Figure 3C), and the degrading efficiencies were higher than those of the single
strain. Although the inoculated cell numbers of strain A1/strain B2 in the combination
degradation were one-half of that in the single strain degradation, the degradation rates
of R-NAP and S-NAP in the combination degradation were 24.8- and 7.5-times that in
the single strain degradation by strain B2 or strain A1, respectively. Additionally, the
degradation rate of Rac-NAP in the combination degradation was 13.4- or 1.2-times
that in the single strain degradation by strain A1 or strain B2, respectively. These data
showed enhanced synergistic catabolism between strains A1 and B2.
4. Proposed metabolic pathways of R-NAP and S-NAP in strain A1 and strain B2
HPLC analysis of samples collected at various times during the degradation of Rac-
NAP, R-NAP or S-NAP by strain A1 or strain B2 showed that the decrease in the
substrates was accompanied by the appearance of new peaks (metabolites) (Figure 4).
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In strain A1, when Rac-NAP was used as the substrate, two peaks (Retention time
(RT)=4.1 min and 4.7 min) gradually increased and then remained unchanged. For R-
NAP, two peaks (RT=4.1 min and 6.4 min) occurred at the beginning, and the peak at
RT=6.4 min finally disappeared. For S-NAP, a peak at RT= 4.7 min (compound 1)
mainly accumulated; a small peak at RT=4.1 min was also found (Figure 4A, B and C).
In strain B2, when Rac-NAP or S-NAP was used as the substrate, two peaks (RT=
7.2 min and 9.4 min) gradually increased, and the peak with RT=7.2 min finally
decreased, showing S-NAP could be transformed to a metabolite (RT=7.2 min) via the
intermediate (RT=9.4 min). For R-NAP, no peak at RT=7.2 min formed, and a peak at
RT=9.4 min was very small and could not be further degraded (Figure 4D, E and F).
Additionally, the metabolite (RT=4.7 min) (compound 1) produced from S-NAP by
strain A1 could be further transformed to an end-product (RT=3.8 min) by strain B2
(Figure S5).
The two metabolites (RT=4.1 min and 6.4 min) produced from R-NAP by strain A1
were identified by HPLC-MS/MS as (R)-N,N-diethyl-2-hydroxypropanamide and
(1R,2S)-1,2-dihydronaphthalene-1,2-diol, with molecular ion peaks [M+H]+ at d 146.11
and 163.06, respectively (Figure 5A, C). The metabolite (RT=4.7 min) was identified
as
(S)-2-(((2S,3S)-2,3-dihydroxy-2,3-dihydronaphthalen-1-yl)oxy)-N,N-
diethylpropanamide at a peak of m/z 304.15 and fragments at m/z 177.06 (loss of
C7H14NO from 304.15) and m/z 159.04 (loss of H2O from 177.06) (Figure 5B).
The metabolite (RT=9.4 min) produced by strain B2 was identified as R-2-(1-
naphthalenyloxy)-propanoic acid or S-2-(1-naphthalenyloxy)-propanoic acid at a peak
of m/z 217.10 and its fragments under ionization conditions were at m/z 171.08 (loss of
CHO2 from 217.10), m/z 143.05 (loss of C2H4 from 171.08) and m/z 127.05 (loss of O
from 143.05) (Figure 5D). The metabolite (RT=7.2 min) was identified as naphthol,
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with a molecular ion peak [M+H]+ at 145.06 and a fragment at m/z 127.05 (loss of HO
from 145.06) (Figure 5E). Another metabolite (RT=3.8 min) was identified as (S)-2-
(((2S,3S)-2,3-dihydroxy-2,3-dihydronaphthalen-1-yl)oxy)propanoic acid with a peak at
m/z 249.07 and a fragment at m/z 177.06 (loss of C3H5O2 from 249.07) (Figure 5F).
According to the results of the HPLC and HPLC-MS/MS analyses, we speculated on
the metabolic pathways of S-NAP and R-NAP in strains A1 and B2. In strain B2, the
amide bond of S-NAP was broken to generate S-2-(1-naphthalenyloxy)-propanoic acid
(S-NP) and diethylamine. The ether bond of S-NP was then cleaved to form α-naphthol,
which finally entered the tricarboxylic acid (TCA) cycle (Figure 6B, green). Meanwhile,
for R-NAP, the amide bond was also broken to generate R-2-(1-naphthalenyloxy)-
propanoic acid (R-NP) and diethylamine, but none of them could be further transformed
(Figure 6A, green). In strain A1, S-NAP slowly transformed to the end-products (S)-2-
(((2S,3S)-2,3-dihydroxy-2,3-dihydronaphthalen-1-yl)oxy)-N,N-diethylpropanamide
and (S)-N,N-diethyl-2-hydroxypropanamide (Figure 6B, orange). Although (S)-2-
(((2S,3S)-2,3-dihydroxy-2,3-dihydronaphthalen-1-yl)oxy)-N,N-diethylpropanamide
could not be further transformed by strain A1, it could be further transformed to
(1R,2S)-1,2-dihydronaphthalene-1,2-diol (RT=3.8 min) by strain B2 (Figure S5). By
contrast, strain A1 could transform R-NAP to (R)-N,N-diethyl-2-hydroxypropanamide
and (1R,2S)-1,2-dihydronaphthalene-1,2-diol, which could be further metabolized to
CO2 and H2O, respectively (Figure 6A, orange).
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5. Structural determination of compound 1 by NMR analysis
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HRTOF-MS analysis of compound 1 (RT=4.7 min) showed a molecular ion peak at
m/z = 304.1578 [M - H]- (Figure 5B), suggesting the molecular formula C17H23NO4,
1
accounting for seven degrees of unsaturation (Figure 7). The H NMR spectrum of
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compound 1 showed three aromatic proton signals in an ABC spin system at δH 7.18
(1H, dd, J = 8.0, 8.0 Hz), 6.82 (1H, d, J = 8.0 Hz), and 6.75 (1H, d, J = 8.0 Hz),
corresponding to a 1,2,3-trisubstituted benzene ring (Figure S6). It also showed
resonances for a disubstituted double bond at δH 6.36 (1H, dd, 9.6, 2.4) and 5.83 (1H,
dt, 9.6, 2.0), and two NCH2CH3 groups at 3.33 (2H, m), 1.05 (3H, t, J = 6.8 Hz), 3.48
(2H, m), and 1.13 (3H, t, J = 6.8 Hz). The resonances at δH 5.05 (1H, d, J = 3.2 Hz) and
4.40 (1H, m) were assigned to the two vicinal oxymethine groups and those at δH 5.16
(1H, q, J = 6.4 Hz) and 1.52 (3H, d, J = 6.4) to the mutually coupled oxymethine and
methyl groups (Table 1), respectively. The 13C NMR data for compound 1 showed the
presence of 17 carbons comprising an amide carbonyl, eight aromatic/olefinic carbons,
and eight aliphatic carbons (Figure S7). The 1H and 13C NMR spectroscopic data were
similar to those of napropamide except for the replacement of the resonances of a
double bond at C-6/C-7 in the napropamide by signals for a vicinal diol in compound 1
(Figure S8, S9). HMBC from H-4 (δH 6.75) to C-6 (δC 63.7) confirmed this assignment
(Figure S10). The relatively small coupling constant (J = 3.2 Hz) between H-6 (δH 5.05)
and H-7 (δH 4.40) suggested that they were oriented on the same side of the cyclohexene
ring. This deduction was supported by an NOE correlation of H-6/H-7 (Figure S11).
Based on these data, compound 1 was identified as 2-((5,6-dihydroxy-5,6-
dihydronaphthalen-1-yl)oxy)-N,N-diethylpropanamide.
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6. Amidohydrolase activity in strain B2
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The metabolic pathways of NAP in strain B2 showed the presence of amidohydrolase.
HPLC results confirmed that the cell-free extract of strain B2 could transform S-
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NAP/R-NAP to S-NP/R-NP (Figure S12). The kinetic parameters and catalytic
efficiency (kcat/Km) of the crude amidohydrolase extracted from strain B2 are
summarized in Table 2. The amidohydrolase from strain B2 showed a catalytic
preference for S-NAP compared with that for R-NAP. It transformed 29% of 0.2 mM
S-NAP within 10 min when 12 μg of cell-free extract was used, while it only
transformed 18% of 0.2 mM R-NAP within 72 h when 600 μg of cell-free extract was
used.
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Discussion
Napropamide (NAP) belongs to the amide herbicide family. It is a chiral herbicide
and among the most commonly used pre-emergence herbicides for fruits, vegetables
and crops to control broadleaf weeds.29 NAP is polar and slightly soluble in water, and
easily passes into the tissues of organisms and soil layer,30 showing a threat to the safety
of drinking water. Although previous investigations have shown that NAP in the
environment was mainly eliminated by microbial transformation, only a few published
studies regarding NAP microbial degradation have been reported and the catabolism of
the different NAP chiral isomers has not been investigated. In this study, we isolated
two Sphingobium species from a Rac-NAP-contaminated soil. Neither strain A1 nor
strain B2 or the combination of strains A1 and B2 could mineralize Rac-NAP, but the
combination of strains A1 and B2 could enhance the degradation of Rac-NAP,
indicating synergistic degradation of the two enantiomers of NAP by strains A1 and B2.
The degradation of R/S-NAP by strain A1 or strain B2 was shown to be
enantioselective. The degradation rates of S/R-NAP by strain A1 and strain B2 were
also different. Under the same inoculum, the degradation rate of R-NAP by strain A1
was 12-times faster than that of S-NAP, while strain B2 completely degraded 0.2 mM
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S-NAP within 7 h and showed no significant degradation of R-NAP. The
enantioselective degradation of the enantiomers might be because of the different
spatial structures of the different enantiomers, causing a different distance between the
catalytic site of the degrading enzymes and target group of enantiomers to be
catalyzed.26 Nevertheless, the specific mechanism involved needs further research. For
example, Wen et al. showed that the main reason for the selectivity of Aspergillus niger
lipase (ANL, EC3.1.1.3) to 2,4-dichlorprop-methyl was enzymatic conformation and
the binding pattern of 2,4-dichlorprop-methyl to this enzyme.31
Strain A1 transformed R-NAP to (R)-N,N-diethyl-2-hydroxypropanamide and
(1R,2S)-1,2-dihydronaphthalene-1,2-diol, and the latter metabolite could be completely
mineralized. However, strain A1 could only transform S-NAP to the end-products (S)-
2-(((2S,3S)-2,3-dihydroxy-2,3-dihydronaphthalen-1-yl)oxy)-N,N-diethylpropanamide
and (S)-N,N-diethyl-2-hydroxypropanamide. Interestingly, strain B2 only transformed
R-NAP to the end-products diethylamine and R-NP but mineralized S-NAP via the
intermediate S-NP. These results showed that strain A1 preferred to degrade R-NAP,
while strain B2 preferred to degrade S-NAP, and strains A1 and B2 complemented each
other to enhance the degradation of both enantiomers. The reason that one single strain
has not evolved the mineralizing pathways for both enantiomers might be that the i)
evolution of complementary pathways for both enantiomers is difficult and needs a long
time; ii) distribution of metabolic pathways in different strains is beneficial to hosts,
and the toxic effects of NAP, as well as its metabolites, are reduced in a single strain;
iii) the degradation efficiency of NAP is enhanced via synergism; and iv) the ecological
balance among the bacterial consortium is maintained.32
During the degradation of R/S-NAP via a single strain A1 or strain B2, end-products
were produced. However, no end-product was detected in the enrichment, suggesting
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that some unisolated strains in the enrichment could degrade these end-products.
Organic pollutants synergistically degraded by bacterial consortia are common.33, 34 For
example, the combination of the Diaphorobacter sp. strain LR2014-1 with
Achromobacter sp. strain ANB-1 resulted in enhanced degradation of the phenylurea
herbicide linuron.35
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412
Notably, the degradation of naphthol, the downstream product of S-NAP, by strain
B2 in MSM was greatly enhanced by the addition of glucose or LB (data not shown).
This phenomenon was similar to that mentioned by Teramoto et al.36 We speculated the
presence of a regulator that can interact with glucose or its metabolite regulates
naphthol degradation in strain B2.
Taken together, we isolated two strains of Sphingobium sp., A1 and B2, which
showed different preferences for the catabolism of the different NAP enantiomers.
Strains A1 and B2 could complement each other to synergistically enhance the
catabolism of both enantiomers. Our study provides new insights into the
enantioselective catabolism of chiral NAP in microorganisms.
Abbreviations
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414
415
416
417
418
419
NAP: Napropamide; Rac-NAP: racemic NAP or Rac-napropamide; TR: thyroxine
receptor; LB: Luria-Bertani medium; MSM: minimal salt medium; Sm: Streptomycin;
HPLC: high-performance liquid chromatography; ECD: electronic circular dichroism;
OD600: optical cell density at 600 nm; HPLC-MS/MS: high-performance liquid
chromatography-tandem mass spectrometry; ESI: electrospray ionization; S-NP: S-2-
(1-naphthalenyloxy)-propanoic acid; R-NP: R-2-(1-naphthalenyloxy)-propanoic acid;
1
1
NMR: nuclear magnetic resonance; H-1H COSY: H-1H correlation spectroscopy;
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HSQC: heteronuclear single quantum coherence; HMBC: heteronuclear multiple bond
correlations; NOESY: nuclear Overhauser effect spectroscopy; RT: retention time; TCA
cycle: tricarboxylic acid cycle; ANL: Aspergillus niger lipase.
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Acknowledgment
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431
This work was supported by the grant from the National Natural Science Foundation of
China (grant no. 31870087) and the Postgraduate Research Practice Innovation
Program of Jiangsu Province (grant no. KYCX17_0658).
Notes
The authors declare no competing financial interest.
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35. Zhang, L.; Hang, P.; Hu, Q.; Chen, X. L.; Zhou, X. Y.; Chen, K.; Jiang, J. D.,
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36. Teramoto, M.; Harayama, S.; Watanabe, K., Phcs represses gratuitous expression
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Figure captions
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Figure 1. UV spectrum scanning of Rac-napropamide (Rac-NAP) after one-week
degradation by the enrichment culture (A) and isolated strain(s) (B). MSM containing
0.2 mM Rac-napropamide and inoculated with sterilized enrichment culture was used
as the control.
Figure 2. Calculated ECD spectra of configurations R-napropamide (red) and S-
napropamide (blue) were compared with the experimental ECD spectra (black) of peak
1 (A) and peak 2 (B) shown in Figure S3.
Figure 3. Degradation of Rac-napropamide (Rac-NAP), R-napropamide (R-NAP) and
S-napropamide (S-NAP) by strain A1 (A), strain B2 (B) and the combination of strains
A1 and B2 (C), respectively. Degradation of R-NAP by strain B2 with a high amount
inoculation (D). Cells of strain A1 or strain B2 were inoculated for individual
degradation in 100 mL of MSM at an initial OD600 value of 0.1 (A and B) and 2.0 (D),
and cells of strain A1 and strain B2 were simultaneously inoculated for a combination
degradation in 100 mL of MSM at an initial OD600 value of 0.05 for every strain (C).
The concentration of Rac-napropamide, R-napropamide and S-napropamide for each
treatment was 0.2 mM. The data are expressed as the mean and standard deviation of
three replicates.
Figure 4. HPLC spectral analysis of the metabolites of Rac-napropamide (A, D), R-
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napropamide (B, E) and S-napropamide (C, F) during the degradation by strain A1 (A,
578
579
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581
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583
584
585
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597
598
B, C) or strain B2 (D, E, F).
Figure 5. HPLC-MS/MS analysis of the metabolites during R-napropamide or S-
napropamide degradation by strain A1 or strain B2. Tandem mass spectrometry of
metabolites at 4.1 min (A), 4.7 min (B) and 6.4 min (C) produced by strain A1 (see in
Figure 4A, 4B and 4C). Tandem mass spectrometry of metabolites at 9.4 min (D), 7.2
min (E) and 3.8 min (F) produced by strain B2 (see Figure 4D, 4E, and 4F and Figure
S4).
Figure 6. Proposed metabolic pathways of R-napropamide (A) and S-napropamide (B)
in strain A1 (in orange) or strain B2 (in green). TCA is an abbreviation for tricarboxylic
acid cycle.
Figure 7. Structure of compound 1.
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Table 1 1H-NMR and 13C-NMR data of compound 1a
NO.
δH
δC
1
2
156.8
114.3
130.0
120.9
135.0
63.7
6.82, d (8.0)
3
7.18, dd (8.0, 8.0)
6.75, d (8.0)
4
5
6
5.05, d (3.2)
7
4.40, m
70.7
8
5.83, dt (9.6, 2.0)
6.36, dd (9.6, 2.4)
133.7
127.0
125.5
73.7
9
10
11
12
13
14
15
16
17
5.16, dd (13.2, 6.4)
1.52, d (6.4)
18.5
170.5
40.7
3.33, m
1.05, t (6.8)
3.48, m
13.0
41.8
1.13, t (6.8)
14.8
600
601
602
603
604
605
606
607
608
609
610
611
612
613
614
615
616
617
618
619
620
621
622
623
a 1H NMR data (δ) were measured in Acetone-d6 at 400 MHz; 13C NMR data (δ) were
measured in Acetone-d6 at 100 MHz.
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Table 2 Kinetic parameters a of crude amidohydrolase extracted from strain B2
Substrate
Specific activity (U/mg)
227.4 ± 3.6
Km (μM)
Vmax (umol/min·mg)
S-napropamide
24.6 ± 1.1
336.7 ± 0.4
R-napropamide
3.2 ± 1.9
32.2 ± 0.3
6.4 ± 0.6
625
626
627
628
629
630
631
632
633
634
635
636
637
638
639
640
641
642
643
644
645
646
647
648
649
650
651
652
653
654
655
656
657
658
659
660
661
662
663
a Kinetic experiments were performed at 30°C in phosphate buffer (50 mM, pH 7.4)
with a final volume of 500 μL. The data are expressed as the mean and standard
deviation of three replicates.
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Figure 1
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666
667
668
669
670
671
672
673
674
675
676
677
678
679
680
681
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Figure 1. UV spectrum scanning of Rac-napropamide (Rac-NAP) after one-week
degradation by the enrichment culture (A) and isolated strain(s) (B). An MSM
containing 0.2 mM Rac-napropamide and inoculated with sterilized enrichment culture
was used as the control.
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Page 29 of 35
Journal of Agricultural and Food Chemistry
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Figure 2
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Figure 2. Calculated ECD spectra of configurations R-napropamide (red) and S-
napropamide (blue) were compared with the experimental ECD spectra (black) of peak
1 (A) and peak 2 (B) shown in Figure S3.
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