Activity Essential Residue Analysis of Taxoid 10β-O-Acetyl Transferase for Enzymatic Synthesis of Baccatin

Abstract

Taxoid 10β-O-acetyl transferase (DBAT) is a key enzyme in the biosynthesis of the famous anticancer drug paclitaxel, which catalyses the formation of baccatin III from 10-deacetylbaccatin III (10-DAB). However, the activity essential residues of the enzyme are still unknown, and the acylation mechanism from its natural substrate 10-deacetylbaccatin III and acetyl CoA to baccatin III remains unclear. In this study, the homology modelling, molecular docking, site-directed mutagenesis, and kinetic parameter determination of the enzyme were carried out. The results showed that the enzyme mutant DBAT resulted in complete loss of enzymatic activity, suggesting that the residue histidine at 162 was essential to DBAT activity. Residues D166 and R363 which were located in the pocket of the enzyme by homology modelling and molecular docking were also important for DBAT activity through the site-directed mutations. Furthermore, four amino acid residues including S31 and D34 from motif SXXD, D372 and G376 from motif DFGWG also played important roles on acylation. This was the first report of the elucidation of the activity essential residues of DBAT, making it possible for the further structural-based re-design of the enzyme for efficient biotransformation of baccatin III and paclitaxel.

Microbial Cell Factory of Baccatin III Preparation in Escherichia coli by Increasing DBAT Thermostability and in vivo Acetyl-CoA Supply

“…There were 13 mutational “hotspots” (Pro37, Val39, Asn42, Ile43, Ser122, His162, His123, Glu124, Ser159, Leu168, Gly171, Ile175, and Ser189) in the solvent channel ( Figure 2 ) chosen for site-directed mutagenesis. As the residue His162 was proved to be the key catalytic site of DBAT in our previous study ( You et al, 2018 ). When the residue His162 was mutated to other amino acids, DBAT will completely lose its activity.…”

“…Since the dbat gene was successfully cloned over two decades, research on DBAT has mainly focused on (i) cloning and heterologous expression of DBAT from different species ( Guo et al, 2007 ; Han et al, 2014 ; Sah et al, 2019 ), (ii) the regional specificity and substrate diversity of DBAT ( Loncaric et al, 2006 , 2007 ), and (iii) the exploration of the unnatural substrate ( Li et al, 2017 ; Lin et al, 2018 ; You et al, 2018 , 2019 ; Huang et al, 2020 ). However, research on the thermal stability of DBAT has not been reported.…”

Omic analysis of the endangered Taxaceae species Pseudotaxus chienii revealed the differences in taxol biosynthesis pathway between Pseudotaxus and Taxus yunnanensis trees

“…In our study, one unigene encoding TS and three unigenes encoding TAT were identified, and they predominantly expressed in T. yunnanensis. DBAT, another rate-limiting enzyme, catalyzes the formation of baccatin III from 10-deacetylbaccatin III [44]. BAPT is responsible for the transfer of a C13-side chain to baccatin III [45].…”

Recent Research Progress in Taxol Biosynthetic Pathway and Acylation Reactions Mediated by Taxus Acyltransferases

“…Li et al [ 72 ] generated a three-dimensional structure of DBAT and identified its active site using alanine scanning, and designed a double DBAT mutant (DBAT G38R/F301V) with a catalytic efficiency approximately six times higher than that of the DBAT wildtype (WT), which improved an in vitro one-pot conversion of 7-b-xylosyl-10-deacetyltaxol to taxol. Moreover, the activity essential residues of the enzyme DBAT, and the acylation mechanism from its natural substrate 10-DAB and acetyl CoA to baccatin III were investigated by You et al [ 74 ]. Among them, residues H162, D166 and R363, located in the catalytic pocket of the enzyme, were important for DBAT activity; and residues S31 and D34 from motif SXXD, D372 and G376 from motif DFGWG were important for acylation.…”