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Engineering of a hybrid enzyme to study thromboxane A2 mediating vascular diseases

Thromboxane A2 (TXA2) is synthesized by a coupling reaction between cyclooxygenase (COX) and TXA2 synthase (TXAS). However, the mechanism of this coordination is poorly understood. To uncover the details of this coupling, it is crucial to have a model showing their coordination. This study focuses on creating a hybrid enzyme to mimic the coupling between COX‐1 and TXAS in the ER membrane. The two enzymes, COX‐1 and TXAS, were joined by a 10 amino acid (aa) linker to create a hybrid enzyme, COX‐1‐10aa‐TXAS. The cDNA of the engineered COX‐1‐10aa‐TXAS was successfully cloned into the pcDNA 3.1 vector containing a cytomegalovirus early promoter and used for gene expression in HEK293 cells. The expression of the hybrid enzyme in the ER membrane, which is similar to that of the co‐expressed COX‐1 and TXAS, was demonstrated by immunostaining. The hybrid enzyme could mimic the triple catalytic functions of the wild type COX‐1 and TXAS, as confirmed by the coupling assays from HPLC and LC/MS as well as Western blot analyses. The results indicated that the biosynthesis of TXA2 is likely through the configuration of the two enzymes in which the orientation of the COX‐1 C‐terminal domain is toward the TXAS N‐terminal domain, and the distance between the two enzymes is likely very short. This study provides a model to understand the molecular mechanism of the coupling between COX‐1 and TXAS in biosynthesis of TXA2, which is involved in strokes.