Cloning And Expression Of The Key Genes Of Prometryn Catabolism In Leucobacter Triazinivorans

Prometryn(4,6-bisisopropylamino-2-methylthio-1,3,5-triazine)is one of the effective broad-spectrum triazine herbicides,which is widely used to control annual grasses and broadleaf weeds in fields such as rice,wheat,and orchards.Due to its stable chemical properties and long residual persistence,prometryn poses a potential threat to ecological environment and human health.Microbial degradation is the main way to remove residual pollution of prometryn in the environment.However,little information on microbial degradation and catabolic mechanism of prometryn was reported in the literature.In the present study,The key genes from the strain Leucobacter triazinivorans JW-1,capable of degrading prometryn,was cloned and expressed for catabolizing prometryn.The molecular mechanism of prometryn catabolism by JW-1 was elucidated by the genes encoding degrading enzymes.The study will provide theoretical guidance and technical support for the bioremediation of triazine herbicides.The main results are as follows:1.The complete genome of Leucobacter triazinivorans JW-1 was sequenced.The potential degrading genes in the strain JW-1 was predicted by reported catabolic pathways of triazine herbicides and bioinformatic analysis.The complete degradation gene cluster was cloned and characterized by heterologous expression and verification of enzyme function.The gene cluster contained three genes,namely,pro A,atz B,and atz C,which could catabolize prometryn to cyanuric acid.The catabolic enzymes ProA,AtzB and AtzC belonged to amidohydrolase family and had a conserved His-Xaa-His amino acid motif.2.Characterization of ProA for catalyzing prometryn was determined.The optimal temperature and p H of ProA were 45? and 7.0,respectively.The enzymatic activity of ProA retained more than 50%after storage at 45? for 1 h,and ProA was stable at p H in a range of 7.0-8.0.The K⁺at 5 mmol/L promoted enzyme activity of ProA,while more than 95%of enzyme activity was inhibited by the addition of 5 mmol/L Fe³⁺or DEPC.The maximum reaction rate of ProA(V_max)for catalyzing prometyn was 0.1?mol/min·mg,and the Michaelis constant(K_m)was 32.6?M.Substrate specificity indicated that ProA could efficiently degrade triazine herbicides containing methylthio group(methoprotryne,desmetryn,and simetryn).However,the low enzyme activity of ProA for catalyzing chlorine-containing triazine herbicides(atrazine)was observed.Based on molecular docking,the catalytic mechanism of different rates of ProA to prometryn and atrazine was revealed from the view of water molecular protonation.3.Characterization of AtzB for catalyzing 2-hydroxypropazine was studied.The optimal temperature and p H for 45? and 9.0 were obtained,respectively.The relative enzyme activity of AtzB remained greater than 70%after storage at 45? for 1 h,and AtzB was stable at p H 8.0-10.0.Both K⁺and Na⁺at 5 mmol/L significantly increased the enzyme activity of AtzB.However,greater than 80%of enzyme activity was significantly inhibited by Cu²⁺and DEPC.The K_mand V_maxof AtzB were 38.4?M and1.0?mol/min/mg,respectively.The determination of substrate specificity showed that AtzB could degrade 2-hydroxypropazine and 2-hydroxyatrazine,indicating that molecular structure of catalytical substrate by AtzB might be required to include a hydroxyl group and one primary or secondary amine substituent on the triazine ring.4.Characterization of AtzC for catalyzing N-isopropylammelide was analyzed.The optimal temperature and p H of AtzC were 35? and 8.0,respectively.The enzyme activity of AtzC remained nearly 70%after storage at 35? within 1 h.The enzyme activity of AtzC was enhanced by metal ions in the presence of K⁺,Ni²⁺,Mg²⁺,Ca²⁺,Mn²⁺,and Co²⁺,but 5 mmol/L of EDTA,a metal-chelator,could almost completely deactivate the enzyme AtzC.The K_mand V_maxof AtzC were 77.4?M and 3.2?mol/min/mg,respectively.The substrate specificity assay showed that AtzC had different degradation activities for 2,4-dihydroxytriazine compounds,such as N-ethylammelide and N-cyclopropylammelide.