The key structure and self-stabilization mechanism of water-soluble interfacial squalene-hopene cyclase

The water insolubility and structural instability of squalene-hopene cyclase (SHC), a membrane-bound interfacial enzyme, pose significant challenges for their use in industrial applications. The membrane-bound nature results in low enzyme yield, cumbersome processes and increased costs. Here, a novel water-soluble catalyst, SaSHC (EC: 5.4.99.17), was discovered from Streptomyces albolongus. This study clearly elucidates its water-soluble structural basis and develop a model for enhancing the water solubility of SHC. The key region, motif 2, contains poly-positively charged amino acids and outward self-anchored structure. The former enhances the electrostatic interaction with the phospholipid head, which makes SaSHC easily dissociated from the cell membrane. And the later ensures the open conformation of the membrane-bound domain. Base on the that, the "outward self-anchored structure dominated-high electrostatic interaction and hydrophobic interaction" (OSA-HELH) model is proposed and applied to optimization SaSHC and AaSHC (from Alicyclobacillus acidocaldarius, tightly bound to the cell membrane). Excitingly, the catalytic efficiency of the SaSHC-L274K was increased by 34.18 %, and mutant AaSHCm2 (AaSHC's motif 2 is replaced by the SaSHC's motif 2) turned into a water-soluble enzyme. In the 100 mL scale-up experiment, the SaSHC-L274K required only 0.04 % Tween 80 to convert 87.82 % squalene, which is an environment-friendly hopene production mode.