An acetophenone carboxylase homolog from Rhodococcus aetherivorans initiates acetone rather than acetophenone catabolism.

Ketones such as acetone and acetophenone are chemically inert and must be activated prior to microbial degradation. In aerobic bacteria such as Rhodococcus, ketone activation is accomplished either by oxygenation or carboxylation reactions. Acetone and acetophenone carboxylases are homologous enzymes, but have different subunit compositions. While acetone carboxylases form heterohexameric complexes out of three distinct subunits, the acetophenone carboxylase core complex contains an additional duplicate of one of these subunits, resulting in a heterooctamer of four different subunits. Moreover, acetophenone carboxylase comprises a detachable fifth subunit ε, which is not present in acetone carboxylases. Here, we show that an apparent acetophenone carboxylase from Rhodococcus aetherivorans initiates acetone rather than acetophenone catabolism. The same strain of R. aetherivorans also degrades acetophenone, but as revealed by proteomics and enzyme activity measurements, this substrate is activated by an oxygenation reaction. In contrast to the canonical enzyme variant, which is restricted to acetone and butanone, the acetone carboxylase from R. aetherivorans apparently also accepts more aliphatic ketones with longer chain length. Furthermore, gene deletion studies confirmed the indispensability of the unique subunit ε for enzyme activity. In conclusion, we identified a novel type of acetone carboxylase with acetophenone carboxylase-like subunit composition and extended substrate spectrum. Phylogenetic analyses suggest that heterodecameric acetone carboxylases are particularly widespread across Actinomycetota and Pseudomonadota.