The transcription factors HNF-4α and NF-κB activate the CDO gene to promote taurine biosynthesis in the golden pompano Trachinotus ovatus (Linnaeus 1758)

Cysteine dioxygenase (CDO) is a rate-limiting enzyme in taurine biosynthesis. Taurine synthesis is limited in marine fish, and most taurine is provided by their diet. Although a nutritional study indicated that the transcription of ToCDO was significantly altered by treatment with 10.5 g/kg taurine in food, the regulatory mechanism of this biosynthesis has not been fully elucidated. In the present study, we identified the sequence features of Trachinotus ovatus cysteine dioxygenase ( ToCDO ), which consists of 201 amino acids. It is characterized by being a member of the cupin superfamily with two conserved cupin motifs located at amino acids 82-102 and 131-145 and with a glutamate residue substituted by a cysteine in its first motif. Moreover, phylogenetic analysis revealed that the similarity of the amino acid sequences between ToCDO and other species ranged from 84.58 % to 91.54 %. Furthermore, a high-performance liquid-phase assay of the activity of recombinantly purified ToCDO protein showed that ToCDO could catalyse the oxidation of cysteine to produce cysteine sulphite. Furthermore, the core promoter region of CDO was identified as- 1182-+1 bp. Mutational analysis revealed that the HNF4 alpha and NF-kappa B kappa B sites significantly and actively affected the transcription of CDO. . To further investigate the binding of these two loci to the CDO promoter, an electrophoretic shift assay (EMSA) was performed to verify that HNF4 alpha-1 and NF-kappa B-1 kappa B-1 interact with the binding sites of the promoter and promote CDO gene expression, respectively. Additionally, cotransfection experiments showed that HNF4 alpha or both HNF4 alpha and NF-kappa B kappa B can significantly influence CDO promoter activity, and HNF4 alpha was the dominant factor. Thus, HNF4 alpha and NF-kappa B kappa B play important roles in CDO expression and may influence taurine biosynthesis within T. ovatus by regulating CDO expression.