Objective To increase the activity of 10-deacetyl baccatin Ⅲ-10β-O-acetyl transferase (DBAT) and the efficiency of in vitro enzymatic synthesis of baccatin Ⅲ.
Method Computer simulation of molecular docking between DBAT and its natural substrate acetyl CoA was performed. Three amino acid residues including C165W, N300I and F160C were chosen for site-directed mutation. The recombinant mutants were expressed in Escherichia coli and their enzymatic properties were determined by reactions in vitro.
Result The relative molecular mass of each of the three mutant enzymes was 67 000, and the optimum reaction temperature was 32.5 ℃ which was consistant with that of the wild type enzyme (DBAT). The optimum reaction pH of DBATF160C or DBAT was 7.5, slightly higher than that of DBATC165W or DBATN300I (pH 7.0). Compared with DBAT, DBATC165W, DBATF160C and DBATN300I had 61.5%, 59.6% and 19.2% higher specific activities, 55.4%、35.1% and 2.9% higher catalytic efficiency, as well as higher Km and vmax.
Conclusion Hydrophobic mutations lead to significantly increased specific activities for mutant enzymes compared with the wild type enzyme. This study provides a basis for efficient in vitro enzymatic synthesis of baccatin Ⅲ, a direct precursor for chemical semi-synthesis of anti-cancer drug paclitaxel.