34 Cyclization Mechanism of Squalene Proposed on the Basis of Site-directed Mutagenesis Experiments

• HOSHINO Tsutomu

  Department of Applied Biological Chemistry, Faculty of Agriculture, Niigata University

• SATO Tsutomu

  Department of Applied Biological Chemistry, Faculty of Agriculture, Niigata University

• Kouda Masanori

  Department of Applied Biological Chemistry, Faculty of Agriculture, Niigata University

• Ohhasi Shumi

  Department of Applied Biological Chemistry, Faculty of Agriculture, Niigata University

• Abe Takamasa

  Department of Applied Biological Chemistry, Faculty of Agriculture, Niigata University

Bibliographic Information

Other Title

• 34 部位特異的変異手法に基づいたスクアレン閉環酵素の触媒反応機構の解析(口頭発表の部)

Abstract

Squalene cyclization mechanism and the active sites of the cyclase are discussed on the basis of the site-directed mutagenesis experiments. 1. DXDDTA motif: the initiation of the polycyclization and stabilization of carobocation intermediate of the initialy formed A-ring through the carboxylate anion of D377. 2. Phe365 stabilizes the C-8 carbocation intermediate of A/B-fused ring system through cation/π interaction. This interaction was confirmed by constructing the mutants F365Y and F365W; these mutants accelerated the reaction velocity and the significant lowering of activation energy for the polycyclization reaction. Tyr420 is also responsible for the formation of B-ring. 3. Phe 601is crucial for the construction of the 6-membered C/D-ring system. The mutant F601A produced the partially cyclized tricyclic 6/6/5-fused13,14 and tetracyclic 6/6/6/5-fused 18. These products could be formed by a Markovnikov closure and indicated the involvements of ring-expansion processes from the 5-membered into the 6-membered ring system for the construction of the C/D-ring system. Compound 18 was also isolated from the mutant W169F and W169H. The presumed carbocationic intermediates 12 and 17 was trapped by using the substrate analogues 27 and 32(34), respectively, with a high nucleophilic hydroxyl group, resulting in the formation of 31 and 35. Thus, we propose that the polycyclization mechanism proceeds via two ring-expansion steps for the formation of C- and D-rings, leading to the formation of anti-Markovnikov adduct 2. 4. The mutant I261A produced a series of carbocationic intermeidates 6/6/5-, 6/6/6/5-, and 6/6/6/6-fused ring systems, giving further insight into the polycycliation mechansim. In the enzymatic products, unnatural natural products are included. 5. Phe 605 would stabilize the cations of 17, 19 and 20 through cation-π interaction, as verified by the isolations of 6/6/6/5- and 6/6/6/6/5-ring skeletons. 6. The methyl at C10-posisiton of 1 is crucial to the correct folding in the active center, this being demonstrated by the substrate analouge 40. The proposed cyclization mechanism has been verified both by the kinetic measurments and by the isolation of the differently cyclized products resulting from each stage of the carbocationic intermediates. Alteration of the cyclase active sites afforded multiple triterpenes in addtion to the understanding of the fundamental issues of squalene cyclization mechanism, suggesting the possibility that we will be able to generate novel triterpenes (unnatural natural products) by rational genetic engineering of squalene cyclase.

Journal

• Symposium on the Chemistry of Natural Products, symposium papers

  Symposium on the Chemistry of Natural Products, symposium papers 41 (0), 199-204, 1999

  Symposium on the Chemistry of Natural Products Steering Committee

Details

• CRID: 1390001206079808128
• NII Article ID: 110006681829
• DOI: 10.24496/tennenyuki.41.0_199
• ISSN: 24331856
• Text Lang: ja
• Data Source:
  • JaLC
  • CiNii Articles
• Abstract License Flag: Disallowed