Laccases can oxidize plenty of substrates by use of molecular oxygen as the final electron acceptor.The broad substrate spectrum is further expanded by using redox mediators in so-called laccase-mediator systems, but the structural studies on interactions between laccases and natural mediators are still absent.In this study, the crystal structure of CotA laccase from Bacillus subtilis 168 has been solved in the presence of sinapic acid (SA), a naturally occurring mediator for laccase.By analyzing CotA/SA complex, a novel substrate binding mode has been revealed.The residue of His419 instead of His497 is bonding to the sinapic acid (SA) as the primary electron acceptor.Moreover, the binding of SA leads to 10 degree rotation on Arg416, our mutagenesis data exhibits that the residue Arg416 is crucial in the oxidation of 2,2-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) and syringaldazine (SGZ).Furthermore, oxidation of several structural analogues of SA was investigated.By analyzing interactions between CotA and SA, it is indicated that the presence of methoxy groups in the ortho-position of the phenolic structure is crucial for the substrate recognition by CotA laccase.This work establishes structure-function relationships for laccase-natural mediator system.Considering the remarkable impact of laccase in various industrial applications, the studies on CotA/SA complex will provide valuable information for developing effective laccasemediator systems with improved biotechnological potential.