Function of N-acetyltransferase in the biotransformation of aniline in the green alga Chlamydomonas reinhardtii

Abstract

Phytoplankton plays a crucial role in the fate of pollutants in aquatic ecosystems by biotransformation and bioaccumulation. Aniline was listed in priority pollutants due to its toxicity and widespread distribution in the aquatic environment. This study focused on investigating the capacity and mechanism of eukaryotic alga Chlamydomonas reinhardtii in transforming aniline. Results showed that the total removal percentage of aniline was 56% within 8 days at an initial concentration of 10 mg · L⁻¹. The percentage of the biotransformation by C. reinhardtii was 23.4%. The biotransformation product was identified as acetanilide, indicating that acetylation was the primary biotransformation pathway. To reveal the key enzyme of the biotransformation process, the N-acetyltransferase (NAT) gene was cloned from the C. reinhardtii genome, and the NAT protein was obtained through heterologous expression. Aniline was significantly transformed by the purified NAT protein in vitro, and the product was also acetanilide. The characteristics of C. reinhardtii NAT in biotransformation of aniline was analyzed by bioinformatics methods. The binding sites in C. reinhardtii NAT for ligands (aniline and acetyl-CoA) were identified. Three highly reserved valine residues (Glu⁸⁵-Asp⁸⁶-Val⁸⁷-Val⁸⁸-Val⁸⁹) as well as GLU¹³¹ and Cys¹²² were the indispensable amino acid residues for the catalysis from aniline to acetanilide. These results demonstrated the capacity of C. reinhardtii in the biotransformation of aniline, and the transformation process was primarily through N-acetylation of aniline to acetanilide catalyzed by NAT enzyme. This study provides novel insights into the biotransformation mechanism of aniline in eukaryotic green alga C. reinhardtii, facilitating the evaluation of the fate of aniline within aquatic ecosystems.