Stig環化酶，如FamC1或HpiC1，能夠催化將(R)-3-geranyl 3-isocyanovinyl indolenine轉化為hapalindole型生物鹼。這些酶催化的過程涉及三個步驟：第一步是香葉基轉移，即Cope重排反應；第二步和第三步是環化的兩階段過程。其中，Cope重排反應是速率決定步驟，但Stig環化酶對於此催化反應的功能仍不明確。本研究中，我們通過分子動力學來平衡FamC1蛋白的結構，並進一步利用量子力學/分子動力學模擬研究Cope重排反應的催化機制。在Cope重排反應過程中，生成的陽離子中間物與苯丙胺酸88發生陽離子-π交互作用，影響了Cope重排反應的途徑和中間物的穩定性。此外，在酶的活性中心，我們觀察到酪胺酸101與反應物之間存在π-π交互作用。我們利用兩種不同方法更進一步地確立本文明確了苯丙胺酸88在Cope重排的重要性。這些關鍵胺基酸在酵素催化中的作用，為設計和開發新的hapalindole型生物鹼提供了重要的指導。;The Stig cyclases, including FamC1 and HpiC1, catalyze the conversion of (R)-3-geranyl-3-isocyanovinylindolenine into hapalindole-type alkaloids through a calcium-dependent enzymatic cascade. This process comprises three key reaction steps: an initial geranyl transfer, followed by a two-stage cyclization. The Cope rearrangement, the rate-determining step, involves the formation of a cationic intermediate. This study used molecular dynamics simulations to equilibrate the FamC1 protein structure, and quantum mechanics/molecular mechanics (QM/MM) simulations were employed to explore the Cope rearrangement mechanism. Our findings indicate that phenylalanine 88 plays a critical role in this step via cation-π interactions, influencing the reaction pathway and intermediate stability. Additionally, π-π interactions between tyrosine 101 and the substrate were observed within the enzyme′s active site. These interactions highlight the importance of specific amino acids in the catalytic function of Stig cyclases, offering insights for the design and development of new hapalindole-type alkaloids.
