Modification Of O-acetyltransferase Based On DBAT Molecular Docking And Its Function

Baccatin ? is an important precursor for the synthesis of paclitaxel,the anticancer drug.The key step of its biosynthesis is the acetylation of the C-10 hydroxyl group of the upstream precursor 10-deacetylbaccatin ? to yield baccatin ?,which is completed by 10-deacetylbaccatin ?-10-?-O-acetyltransferase(DBAT)with substrate acetyl-Co A.However,the enzyme activity of natural DBAT is relatively low,looking for an O-acetyltransferase that can replace DBAT is expected to increase the yield of the bacatein ?.In this study,the homoserine O-acetyltransferase gene and the maltose O-acetyltransferase gene were cloned from Pleurotus ostreatus and expressed in prokaryotic expression,at the same time,the molecular docking transformation of DBAT was carried out on the homoserine O-acetyltransferase P1-HTA,and it was explored whether the mutase could replace DBAT to generate bacatein ?.The main findings of this paper are as follows:(1)Tow homoserine O-acetyltransferase genes(p1-hta?p2-hta1)and a maltose O-acetyltransferase gene(p-mta1)were cloned from pleurotus ostreatus by reverse transcription PCR.(2)The expression vector p ET32a-p1-hta containing gene p1-hta constructed by restriction enzymes was transformed into E.coli BL21.The expression of soluble protein reached the highest level in the engineered strain BL21/p ET32a-p1-hta after 16 h induction with 0.2m M IPTG in 25?.The expression vectors p ET32a-p1-hta and p ET22b-p1-hta were subsequently transformed into E.coli engineered strains Rosetta,Origami B and Rosetta gami plys S,respectively,to investigate the expression of the enzyme in different combinations of vectors and hosts,results showed that the expression of soluble protein reached the higher level with engineering strain BL21/p ET32a-p1-hta?Rosetta/p ET32a-p1-hta and Origami B/p ET32a-p1-hta.(3)The enzyme activities and enzymatic properties of P1-HTA,P2-HTA1 and P2-HTA were tested.Results showed that the P1-HTA has a specific activity of 15.323±0.183m U·mg^-1,K_m was 0.087±0.033?M,k_cat/K_m was 19.828;P2-HTA1 has a specific activity of9.839±0.029 m U·mg^-1,K_m was 0.125±0.037?M,k_cat/K_m was 13.488;P2-HTA has a specific activity of 10.034±0.012 m U·mg^-1,K_m was 0.117±0.012?M,k_cat/K_m was 15.786.Explored whether P1-HTA,P2-HTA1,P2-HTA,P-MTA1 and P-MTA could replace DBAT as an alternative enzyme to generate baccatin ?,the results were not.(4)The mutant enzyme P1-HTA-MUT was obtained through the computer simulation docking of 10-DAB with DBAT or P1-HTA,including the active pocket in P1-HTA was replaced by the corresponding substrate binding pocket in DBAT with 10-DAB.The enzyme activities and enzymatic properties of mutant enzyme P1-HTA-MUT were tested.Results showed that the P1-HTA-MUT has a specific activity of 15.323±0.183m U·mg^-1,which was improved 1.4 times compared with wild-type P1-HTA;the K_m of P1-HTA-MUT was 0.076±0.008?M,the affinity of substrate with enzyme was increased about 0.011?M compared with wild type P1-HTA;the k_cat/K_m of P1-HTA-MUT was28.737,the catalytic efficiency of the enzyme was increased by about 1.4 times compared to the wild type P1-HTA.The mutant enzyme P1-HTA-MUT still was not replaced DBAT as an alternative enzyme to generate baccatin ?.In this study,the baccatin ? could not be generated with the co-substrate 10-DAB and acetyl-Co A under the catalysis of P1-HTA-MUT,indicating that the single replacement of the all active pocket could not change the substrate specificity of the enzyme,and the effects of electrostatic interaction,hydrogen bonding,hydrophobic interaction and steric hindrance should be considered in the mutation modification with enzyme.However,when the co-substrate were homoserine and acetyl-Co A,the specific activity of P1-HTA-MUT was improved to generate homoserine O-acetyltransferase,which is a important precursor for the synthesis of methionine.Therefore,the enzyme activity is improved by modification could provide a reference for the synthesis of methionine.