Critical role of the trans-scirpusin A Hydroxylase (SAH), A Cyt P450 enzyme, involved in trans-scirpusin A biosynthesis in Vitis vinifera

Abstract

Scirpusin A is generally present in plants in trans configuration, which has strong biological activities, but the synthetic pathway of trans-scirpusin A still unclear. We previously identified three candidate sequences in the transcriptome of Vitis vinifera treated by UV-B, which may be involved in the synthesis of trans-scirpusin A. Here, we characterized a trans-scirpusin A hydroxylases (SAH) gene in Vitis vinifera. The SAH ORF is predicted to encode 278 amino acids belonging to Cytochrome P450 proteins (CYP) super family. In addition, molecular simulation was performed that the binding site between trans-ε-viniferin and SAH is Ser 276 in SAH, and it was speculated that trans-ε-viniferin and SAH may be combined by hydrogen interaction. Further, the function of the candidate gene was tested using heterologous expression in Pichia pastoris. UHPLC-ESI-MS2 analysis revealed that SAH can catalyze trans-ε-viniferin to trans-scirpusin A in vitro. And subcellular analysis showed that the SAH gene was expressed in the nucleus and membrane. Overall, our research demonstrates that SAH is trans-scirpusin A enzyme in trans-scirpusin A biosynthesis and provides a new sight for exploring the biosynthesis mechanism of trans-scirpusin A.