Functional Study Of Two Types Of Sex Pheromone-degrading Enzymes In Plutella Xylostell

The diamondback moth（DBM）,Plutella xylostella（L.）（Lepidoptera:Plutellidae）,is one of the world’s most important pests of cruciferous vegetables.The planting area of cruciferous crops such as cabbage and cauliflower in China ranks first globally,providing sufficient food sources for diamondback moths.However,the long-term dependence on chemical pesticide control has made the diamondback moth one of the pests with the most serious pesticide resistance.Thus,novel green control methods are urgently needed.Insects have an elaborate olfactory system that plays a vital role in detecting odorants in the environment to facilitate aggregation behaviors and locate suitable mates,hosts,and oviposition sites.The genes of insect perception of odorant have been widely studied,but the molecular mechanism of odor signal termination is less studied.Several mechanisms suggest that enzymatic inactivation could contribute to the signal termination process,such as odorant-degrading enzymes（ODE）.To date,a few ODE genes have been identified and characterized in detail in insect herbivores.Some members of carboxylesterase（CCE）and aldehyde oxidase（AOX）are potential ODE genes responsible for degrading ester and aldehyde odorant molecules,respectively.CCE and AOX are two major metabolic enzymes that oxidize a variety of esters and aromatic aldehydes,respectively,and they may also play a significant role in the detoxification and degradation of environmental chemical odorants.In this study,two paralogous antennae-enriched CCEs（PxylCCE16a and PxylCCE16c）and one PxylAOX3 from Plutella xylostella were identified and functionally characterized.First,high-purity of active recombinant PxylCCE16a,16c and PxylAOX3 proteins have been obtained from Sf9 insect cells by Ni²⁺affinity purification.For the two sex pheromone components,antennal extracts displayed degradation rates against Z9-14:OAc and Z11-16:OAc of 25.44±1.99%and 24.93±2.50%,respectively,while PxylCCE016c presented degradation rates of 27.64±0.79%and 24.40±3.07%,respectively.However,PxylCCE016a had relatively low activities at only 8.40±0.27%and 6.87±0.60%,respectively.For plant volatiles,the two CCE and antennal extracts all have a high degradation rate on Z3-6:OAc among five tested esters,at 70.90±1.44%（antennal extracts）,14.83±0.76%（PxylCCE016a）,and 63.51±2.30%（PxylCCE016c）.Interestingly,only PxylCCE016c can be inhibited by a common esterase inhibitor triphenyl phosphate（TPP）with LC₅₀ of 1570±520μmol L^–1.In addition,PxylAOX3 displayed an extensive degradation zymogram that oxidized not only major sex pheromone component Z11-16:Ald and analogs（Z11-14:Ald,Z9-16:Ald,and Z10-16:Ald）with a relative degradation rate more than 40%,but also effectively oxidized plant-derived aldehydes and xenobiotics（at least 24.96%for aldicarb to the control substrate 100%phenylacetaldehyde）.In addition,the enzymatic activity analysis of PxylAOX3 at different temperatures showed that the thermal stability of PxylAOX3 gradually decreased with increasing temperature.The oxidation activity of PxylAOX3 was relatively stable during 20–40°C and started to decline at50°C slightly,with 83.62±2.44%activity remaining.The enzyme activity plummeted sharply from 50 to 70°C,with only 64.20±2.50%and 23.28±1.56%activities at 60and 70°C,respectively.Furthermore,the activity of the two newly identified CCEs and the PxylAOX are pH-dependent.The activity at pH 6.5 is significantly higher than that at pH 5.0.Thus,the enzyme activity assays of PxylAOX3 were conducted under various pH conditions ranging from 5.0 to 8.0.PxylAOX3 possesses the optimum activity at the pH value of8.0 and relatively high activity at 7.0 and 7.5,approximately 80%compared to 8.0.Additionally,a higher pH（>8.0）resulted in the rapid nonenzymatic reduction of MTT that inhibited the AOX activity measurement due to an unacceptable background absorbance.Furthermore,low activity was detected at pH 6.0（22.47±2.48%）,and no activity was detected at pH 5.0.The results showed that the enzyme activities soared when the pH shifted from 5.0 to 7.0 in both PxylCCE016a and 016c regardless of whether the sex pheromone component（Z11-16:OAc）or the typical plant volatile（Z3-6:OAc）was the substrate,which is comparable to other moth ODEs.For example,at pH=5.0,the degradation activities of PxylCCE016a on sex pheromone component Z11-16:OAc and typical plant volatiles Z3-6:OAc were only about 5.0%and 0.4%,respectively.The degradation activities of Pxy CCE016c to these two substrates are also only about 7.0%and 3%,respectively.At pH=6.5,the activities of PxylCCE016a against these two substrates were 7.0%and 2.0%,respectively.The activities of Pxy CCE016c against these two substrates were 9.0%and 19%,respectively.Furthermore,homology modeling and substrate docking were constructed to predict PxylAOX3,PxylCCE016a and 016c 3D structures,potential active binding sites,and amino acid residues to facilitate further development design of pesticides or inhibitors.In this study,PxylCCE016a and Pxy CCE016c present high-matched 3D structures with similarα-helixes andβ-loops and possess the same enzyme active center（GESAG）motif and catalytic triad（Ser186-Glu320-His442 and Ser186-Glu321-His444）.The key amino acid residues they mainly bind are PHE-325,SER-390,GLN-277,TRP-219,and MET-391.The PxylAOX3 3D structure showed that it possesses the conservative domains of the AOX family,such as the flavin-containing region（FAD-binding domain）and two putative iron-sulfur（2Fe-2S）redox center,molybdenum cofactor-binding site,such as Gln744,Tyr855,Pro970,Tyr974,Phe969,and Glu1214.This study identified and functionally analyzed the enzymatic properties of ester and aldehyde sex pheromone-degrading enzymes of Plutella xylostella,which provided a theoretical basis for further analysis of the molecular sensing mechanism of moth sex pheromone recognition.Moreover,the current study provides novel potential pesticide targets for the notorious moth Plutella xylostella.