Bidirectional and regiospecific C21-modification of steroids by a chloramphenicol O-acetyltransferase.

Bidirectional acetylation and deacetylation of small molecules by acetyltransferases (ATs) remain poorly understood. In this study, we present the structural, functional, and computational characterization of a chloramphenicol O-AT from Bacillus sp. PAMC22265 (AT65) that exhibits bidirectional catalytic activity toward the regiospecific C21-acetylation and deacetylation of steroid substrates. The crystal structure of AT65 was determined at 2.40 Å resolution (PDB ID: 24ZY), revealing a trimeric architecture in which the putative active-sites located at the interfaces between adjacent subunits. In vitro kinetic analyses of both acetylation and deacetylation reactions demonstrated substrate-dependent catalytic efficiencies, with comparable activity under the experimental conditions. Molecular docking, 100 ns molecular dynamics simulations, hydrogen-bond analysis, and MM-PBSA free-energy calculations supported stable substrate binding within the active site and suggested that His187 and Asp191 may contribute to substrate recognition and catalysis. Consistent with this observation, substitution of His187 with alanine abolished detectable enzymatic activity, highlighting its functional importance. Whole-cell biotransformation further demonstrated the selective production of C21-acetylated steroid derivatives, some of which exhibited preliminary antiproliferative activity against murine breast cancer cell lines. Collectively, these findings provide structural, biochemical, and computational insights into the bidirectional catalytic properties of AT65 and establish a foundation for further mechanistic studies and the development of selective enzymatic strategies for steroid modification.