The biochemical properties of a novel paraoxonase-like enzyme in Trichoderma atroviride strain T23 involved in the degradation of 2,2-dichlorovinyl dimethyl phosphate

Dichlorvos, is a broad-spectrum organophosphorus pesticide that is widely applied in the agricultural industry and considered a pollutant to fish and bees. T. atroviride strain T23, an efficient DDVP-degrading strain, could convert DDVP to dichloroacetic acid, 2,2-dichloroethanol and phosphoric acid through mineralization. RT-qPCR analysis showed TaPon1-like encoding an organophosphorus hydrolase, is continuously highly expressed in the process of degrading DDVP. TaPon1-like contained an open reading frame of 1317 bp, and the deduced amino acid sequence shared 21% homology with HuPON1, which also exhibits excellent hydrolysis of organophosphate-oxons compounds. By analysis of gene knockout, we found the ΔTaPon1-like knockout strain KO1 lost 35.6% of its DDVP-degradation capacity at 24 h, but this loss of degradation activity was recovered when the gene was complemented. Furthermore, the purified recombinant protein reTAPON1-LIKE, could transform DDVP only to dimethyl phosphate and showed significant paraoxonase activity (1028 U L−1). The reTAPON1-LIKE enzyme showed a broad degradation spectrum, degrading not only DDVP but also organophosphate-oxons and lactone. The kinetic parameters (Km and kcat) of the purified reTAPON1-LIKE were determined to be 0.23 mM and 204.3 s−1 for DDVP, respectively. The highest activity was obtained at 35 °C, and the optimal pH was 8.5. The activity of reTAPON1-LIKE was enhanced most significantly when 1.0 mM Ca2+ was added but declined when 1.0 mM Cu2+ was added. These results showed TAPON1-LIKE play an important role for DDVP degradation in the first step by T23 and provided clue to comprehensively understanding the degradation mechanism of organophosphate-oxons pesticides by filamentous fungi. Importance The large amounts of residues of organophosphate pesticides in agroecological system has become a great threat to the safety of environment and humans. Bioremediation in association with microbial is innovative technology having a potential to alleviate such pollution problems. The genus Trichoderma is genetically diverse with capabilities to degrade chemical pesticides among different strains with agricultural significance. As a typical organophosphorus pesticide, it is one of the most employed compounds of the family. Though it was classified as a highly toxic pesticide by WHO due to its hazardous properties, it plays an important role in the control of plant pests, food storage and homes, as well as to treat infections in livestock. Therefore, we use DDVP as a model of organophosphate pesticide to study the mechanism of Trichoderma degrading organophosphate pesticides, for the aim of globally understanding molecular mechanism of enzymatic degradation of organophosphate pesticides by beneficial fungi.

contained an open reading frame of 1317 bp, and the deduced amino acid sequence 23 shared 21% homology with HuPON1, which also exhibits excellent hydrolysis of 24 organophosphate-oxons compounds. By analysis of gene knockout, we found the 25 ΔTaPon1-like knockout strain KO1 lost 35.6% of its DDVP-degradation capacity at 24 h, 26 but this loss of degradation activity was recovered when the gene was 27 complemented. Furthermore, the purified recombinant protein reTAPON1-LIKE, 28 could transform DDVP only to dimethyl phosphate and showed significant 29 paraoxonase activity (1028 U L -1 ). The reTAPON1-LIKE enzyme showed a broad 30 degradation spectrum, degrading not only DDVP but also organophosphate-oxons 31 and lactone. The kinetic parameters (K m and k cat ) of the purified reTAPON1-LIKE were 32 determined to be 0.23 mM and 204.3 s -1 for DDVP, respectively. The highest activity 33 was obtained at 35 °C, and the optimal pH was 8.5. The activity of reTAPON1-LIKE 34 was enhanced most significantly when 1.0 mM Ca 2+ was added but declined when   Gene expression analysis using a reverse transcription quantitative polymerase chain 187 reaction (RT-qPCR) revealed a significant (P < 0.001) induction of TaPon1-like with 188 different DDVP concentrations at 24 h (Fig. 3A). In addition, expression of TaPon1-like 189 was strongly induced (P < 0.001) when T23 was exposed to 300 μg mL -1 DDVP. As   HuPON1 is a six-blade β-propeller containing two calcium ions in a central tunnel.

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The tunnel-buried calcium is critical for the enzyme's conformational stability, and 215 the solvent-exposed calcium residing at the bottom of the active-site cavity is needed 216 for catalysis (14). In comparison with HuPON1, the mimetic TAPON1-LIKE contained 217 Asn168, which ligates the catalytic calcium, while the Asp183-His184 dyad is vital for 218 stabilizing the catalytic calcium ion.

219
Moreover, TAPON1-LIKE was found to have a significant motif, XXXTLVDNXXXXD, 221 which may be the direct catalytic calcium metal-binding region for activating 222 hydrolysis of OPPs, which interact with the side chains of Asp269 and Glu53. Motifs 223 PXXPXXIXLMD or DXXXXXXXXMYLXVVN may be another metal-binding regions for 224 supposed catalytic calcium ion (15) ( Fig. 5 and Fig. S4C).  TaPon1-like transcript in six transformants, while an   244 amplification product of the desired size was found in the WT (Fig. S2D). Southern 245 blotting ( Fig. S2F) results showed TaPon1-like was replaced using an 800 bp fragment 246 of hygB. We selected one suitable deletion transformant designated KO1. Using 247 similar methods, TaPon1-like complementation transformants were screened, and 248 the suitable one was designated CO1 (Fig. S3).

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The DDVP degradation rates of strain T23 and related mutants were measured by 251 GC-FPD at an initial concentration of 300 μg mL -1 in Burk medium for 168 hours (h).

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Strain T23 exhibited an excellent efficiency of degrading DDVP, and the degradation 253 rate value was 56% at the first 24 h (Fig. 6   produced a single band and was designated reTAPONN1-LIKE (Fig. 7).

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The DDVP degradation products of purified reTAPON1-LIKE were evaluated by GC-MS 288 analysis. Only tert-butyldimethylsilyl derivative of dimethyl phosphate, with a 289 retention time of 14.106 min, was detected as a metabolite of DDVP biodegradation 290 (Fig. S5). Therefore, we confirmed that TAPON1-LIKE is the enzyme to transform 291 DDVP into dimethyl phosphate.  A variety of environmental factors such as pH and temperature exert varied effects 342 on the enzyme activity and stability (6). The reTAPON1-LIKE protein exhibited high 343 activity to DDVP between pH 6.5 and 10.0 (more than 50% relative activity), and pH 344 8.5 was optimum for its activity (Fig. 8A). Serum paraoxonase 1 activity was reported 345 to occur from pH 7 to 10.5 (25), and the ideal pH of the reaction is in the range of 8 346 to 8.5 (26, 27).

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The reTAPON1-LIKE protein was active at 20-50 °C, with an optimum temperature of 349 35 °C using DDVP as substrate (Fig. 8B). An increase in temperature of 1 °C was 350 associated with a 4.5% increase in PON1 activity when phenyl acetate was used as 351 substrate (28). The optimum temperature of hydrolysis for paraoxon and phenyl acetate ranges from 30-45 °C (29). The TAPON1-LIKE amino acid sequence shared identity with the sequences of the 357 metal-dependent hydrolases; therefore, it was deduced that metal ions might also 358 affect reTAPON1-LIKE enzymatic activity. As shown in Table 2 As can be seen in Table 2 The strains, plasmids, and primers used in this study are listed in Table 3 and Table 4.  Gene expression analysis of TaPon1-like genes in strain T23 was performed under the 541 two different conditions described above. RNA extraction was performed using the 542 Qiagen RNeasy kit following the manufacturer's protocol (Qiagen, Hilden, Germany).

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One microgram of total RNA was reverse transcribed in a total volume of 20 μL as Strain T23 (500 μg mL -1 ) was inoculated into a 500 mL Erlenmeyer flask containing 556 200 mL of Burk medium, and the culture was cultivated as described above. The 557 metabolites were analyzed by gas chromatography-mass spectrometry (GC-MS).

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After an incubation period, the mycelia were removed by filtering through filter 559 paper. Then, 2% NaCl and 2 mL of HCl were added; 100 mL of anhydrous ethyl ether 560 was subsequently added (equal to 50% of the total volume), and the sample was

938
DDVP was used as the substrate. Activity at the optimal pH and temperature was 939 defined as 100%. Data are expressed as the means ± standard errors of three 940 replicates.

Mutant complementation
TaPon1-like-PRO-F AAACGACGGCCAGTGCCAAGCTTTTAGCTCAAAGCCCAGAAGCA TaPon1-like-PRO-R  TGATTGAGGCGCGGGCCGCCATCGTGACGAATACGCAAGCAC  TaPon1-like-TER-F  TTGCTGTCAAGATCGATTTGATGGTGAGCAAGGGCGAGGA  TaPon1-like-TER-R  GAGCTCGGTACCCGGGGATCCAACCCAGGGGCTGGTGACGG  G418-F  GTTGTCACTGAAGCGGGAAGG         Activity at the optimal pH and temperature was defined as 100%. Data are expressed as the means ± standard errors of three replicates.