Cation- Interaction in the Polyolefin Cyclization Cascade Uncovered by Incorporating Unnatural Amino Acids into the Catalytic Sites of
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- 作者：Noriko Morikubo ; Yoriyuki Fukuda ; Kazumasa Ohtake ; Naoko Shinya ; Daisuke Kiga ; Kensaku Sakamoto ; Miwako Asanuma ; Hiroshi Hirota ; Shigeyuki Yokoyama ; Tsutomu Hoshino
- 刊名：Journal of the American Chemical Society
- 出版年：2006
- 出版时间：October 11, 2006
- 年：2006
- 卷：128
- 期：40
- 页码：13184 - 13194
- 全文大小：283K
- 年卷期：v.128,no.40(October 11, 2006)
- ISSN：1520-5126
文摘
It has been assumed that the 
-electrons of aromatic residues in the catalytic sites of triterpenecyclases stabilize the cationic intermediates formed during the polycyclization cascade of squalene oroxidosqualene, but no definitive experimental evidence has been given. To validate this cation- interaction,natural and unnatural aromatic amino acids were site-specifically incorporated into squalene-hopene cyclase(SHC) from Alicyclobacillus acidocaldarius and the kinetic data of the mutants were compared with that ofthe wild-type SHC. The catalytic sites of Phe365 and Phe605 were substituted with O-methyltyrosine,tyrosine, and tryptophan, which have higher cation- binding energies than phenylalanine. Thesereplacements actually increased the SHC activity at low temperature, but decreased the activity at hightemperature, as compared with the wild-type SHC. This decreased activity is due to the disorganization ofthe protein architecture caused by the introduction of the amino acids more bulky than phenylalanine.Then, mono-, di-, and trifluorophenylalanines were incorporated at positions 365 and 605; these aminoacids reduce cation- binding energies but have van der Waals radii similar to that of phenylalanine. Theactivities of the SHC variants with fluorophenylalanines were found to be inversely proportional to the numberof the fluorine atoms on the aromatic ring and clearly correlated with the cation- binding energies of thering moiety. No serious structural alteration was observed for these variants even at high temperature.These results unambiguously show that the -electron density of residues 365 and 605 has a crucial rolefor the efficient polycyclization reaction by SHC. This is the first report to demonstrate experimentally theinvolvement of cation- interaction in triterpene biosynthesis.
-electrons of aromatic residues in the catalytic sites of triterpenecyclases stabilize the cationic intermediates formed during the polycyclization cascade of squalene oroxidosqualene, but no definitive experimental evidence has been given. To validate this cation- interaction,natural and unnatural aromatic amino acids were site-specifically incorporated into squalene-hopene cyclase(SHC) from Alicyclobacillus acidocaldarius and the kinetic data of the mutants were compared with that ofthe wild-type SHC. The catalytic sites of Phe365 and Phe605 were substituted with O-methyltyrosine,tyrosine, and tryptophan, which have higher cation- binding energies than phenylalanine. Thesereplacements actually increased the SHC activity at low temperature, but decreased the activity at hightemperature, as compared with the wild-type SHC. This decreased activity is due to the disorganization ofthe protein architecture caused by the introduction of the amino acids more bulky than phenylalanine.Then, mono-, di-, and trifluorophenylalanines were incorporated at positions 365 and 605; these aminoacids reduce cation- binding energies but have van der Waals radii similar to that of phenylalanine. Theactivities of the SHC variants with fluorophenylalanines were found to be inversely proportional to the numberof the fluorine atoms on the aromatic ring and clearly correlated with the cation- binding energies of thering moiety. No serious structural alteration was observed for these variants even at high temperature.These results unambiguously show that the -electron density of residues 365 and 605 has a crucial rolefor the efficient polycyclization reaction by SHC. This is the first report to demonstrate experimentally theinvolvement of cation- interaction in triterpene biosynthesis.