 Abstract di-tepine synthase-vena is responsible for assembling Venezueline with a unique 5-5-6-7 tetracyclic skeleton from geronil-geronil pyrophosphate. It also accepts geronil-pyrophosphate and pharnasyl-pyrophosphate as alternative substrates. The crystal structures of vena in both apo-form and holo-form in complex, with a trinuclear magnesium cluster and pyrophosphate group have been reported. Functional and structural investigations on the atypical 115-DSFVSD120 motif of vena, versus the canonical aspirate motif of DDXXXDE, revealed that the absence second-asp of canonical motif is functionally replaced by Ser116 and GLN83. Multiscale computational simulations and structure-directed mutagenesis provided significant mechanistic insights into the substrate selectivity and catalytic promiscuity of vena. Furthermore, vena was semi-rationally engineered into a cester-terpene synthase to recognize the larger substrate geronil-pharnasyl pyrophosphate. This article was authored by Zhong Li, Lilan Zhong, Kang Wei Shu, and others. We are article.tv, links in the description below.