Cation-π interaction in the polyolefin cyclization cascade uncovered by incorporating unnatural amino acids into the catalytic sites of squalene cyclase

Noriko Morikubo, Yoriyuki Fukuda, Kazumasa Ohtake, Naoko Shinya, Daisuke Kiga, Kensaku Sakamoto, Miwako Asanuma, Hiroshi Hirota, Shigeyuki Yokoyama, Tsutomu Hoshino

Research output: Contribution to journalArticle

56 Citations (Scopus)

Abstract

It has been assumed that the π-electrons of aromatic residues in the catalytic sites of triterpene cyclases stabilize the cationic intermediates formed during the polycyclization cascade of squalene or oxidosqualene, but no definitive experimental evidence has been given. To validate this cation-π 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 of the wild-type SHC. The catalytic sites of Phe365 and Phe605 were substituted with O-methyltyrosine, tyrosine, and tryptophan, which have higher cation-π binding energies than phenylalanine. These replacements actually increased the SHC activity at low temperature, but decreased the activity at high temperature, as compared with the wild-type SHC. This decreased activity is due to the disorganization of the 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 amino acids reduce cation-π binding energies but have van der Waals radii similar to that of phenylalanine. The activities of the SHC variants with fluorophenylalanines were found to be inversely proportional to the number of the fluorine atoms on the aromatic ring and clearly correlated with the cation-π binding energies of the ring moiety. No serious structural alteration was observed for these variants even at high temperature. These results unambiguously show that the π-electron density of residues 365 and 605 has a crucial role for the efficient polycyclization reaction by SHC. This is the first report to demonstrate experimentally the involvement of cation-π interaction in triterpene biosynthesis.

Original languageEnglish
Pages (from-to)13184-13194
Number of pages11
JournalJournal of the American Chemical Society
Volume128
Issue number40
DOIs
Publication statusPublished - 2006 Oct 11
Externally publishedYes

Fingerprint

Cyclization
Polyolefins
Cations
Amino acids
Catalytic Domain
Positive ions
Amino Acids
Binding energy
Phenylalanine
Triterpenes
Temperature
Alicyclobacillus
Electrons
Squalene
Biosynthesis
Aromatic Amino Acids
Carboxylic acids
Fluorine
Carrier concentration
Tryptophan

ASJC Scopus subject areas

  • Chemistry(all)

Cite this

Cation-π interaction in the polyolefin cyclization cascade uncovered by incorporating unnatural amino acids into the catalytic sites of squalene cyclase. / Morikubo, Noriko; Fukuda, Yoriyuki; Ohtake, Kazumasa; Shinya, Naoko; Kiga, Daisuke; Sakamoto, Kensaku; Asanuma, Miwako; Hirota, Hiroshi; Yokoyama, Shigeyuki; Hoshino, Tsutomu.

In: Journal of the American Chemical Society, Vol. 128, No. 40, 11.10.2006, p. 13184-13194.

Research output: Contribution to journalArticle

Morikubo, Noriko ; Fukuda, Yoriyuki ; Ohtake, Kazumasa ; Shinya, Naoko ; Kiga, Daisuke ; Sakamoto, Kensaku ; Asanuma, Miwako ; Hirota, Hiroshi ; Yokoyama, Shigeyuki ; Hoshino, Tsutomu. / Cation-π interaction in the polyolefin cyclization cascade uncovered by incorporating unnatural amino acids into the catalytic sites of squalene cyclase. In: Journal of the American Chemical Society. 2006 ; Vol. 128, No. 40. pp. 13184-13194.
@article{4514740d6892463b9f2457d675d58fcd,
title = "Cation-π interaction in the polyolefin cyclization cascade uncovered by incorporating unnatural amino acids into the catalytic sites of squalene cyclase",
abstract = "It has been assumed that the π-electrons of aromatic residues in the catalytic sites of triterpene cyclases stabilize the cationic intermediates formed during the polycyclization cascade of squalene or oxidosqualene, but no definitive experimental evidence has been given. To validate this cation-π 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 of the wild-type SHC. The catalytic sites of Phe365 and Phe605 were substituted with O-methyltyrosine, tyrosine, and tryptophan, which have higher cation-π binding energies than phenylalanine. These replacements actually increased the SHC activity at low temperature, but decreased the activity at high temperature, as compared with the wild-type SHC. This decreased activity is due to the disorganization of the 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 amino acids reduce cation-π binding energies but have van der Waals radii similar to that of phenylalanine. The activities of the SHC variants with fluorophenylalanines were found to be inversely proportional to the number of the fluorine atoms on the aromatic ring and clearly correlated with the cation-π binding energies of the ring moiety. No serious structural alteration was observed for these variants even at high temperature. These results unambiguously show that the π-electron density of residues 365 and 605 has a crucial role for the efficient polycyclization reaction by SHC. This is the first report to demonstrate experimentally the involvement of cation-π interaction in triterpene biosynthesis.",
author = "Noriko Morikubo and Yoriyuki Fukuda and Kazumasa Ohtake and Naoko Shinya and Daisuke Kiga and Kensaku Sakamoto and Miwako Asanuma and Hiroshi Hirota and Shigeyuki Yokoyama and Tsutomu Hoshino",
year = "2006",
month = "10",
day = "11",
doi = "10.1021/ja063358p",
language = "English",
volume = "128",
pages = "13184--13194",
journal = "Journal of the American Chemical Society",
issn = "0002-7863",
publisher = "American Chemical Society",
number = "40",

}

TY - JOUR

T1 - Cation-π interaction in the polyolefin cyclization cascade uncovered by incorporating unnatural amino acids into the catalytic sites of squalene cyclase

AU - Morikubo, Noriko

AU - Fukuda, Yoriyuki

AU - Ohtake, Kazumasa

AU - Shinya, Naoko

AU - Kiga, Daisuke

AU - Sakamoto, Kensaku

AU - Asanuma, Miwako

AU - Hirota, Hiroshi

AU - Yokoyama, Shigeyuki

AU - Hoshino, Tsutomu

PY - 2006/10/11

Y1 - 2006/10/11

N2 - It has been assumed that the π-electrons of aromatic residues in the catalytic sites of triterpene cyclases stabilize the cationic intermediates formed during the polycyclization cascade of squalene or oxidosqualene, but no definitive experimental evidence has been given. To validate this cation-π 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 of the wild-type SHC. The catalytic sites of Phe365 and Phe605 were substituted with O-methyltyrosine, tyrosine, and tryptophan, which have higher cation-π binding energies than phenylalanine. These replacements actually increased the SHC activity at low temperature, but decreased the activity at high temperature, as compared with the wild-type SHC. This decreased activity is due to the disorganization of the 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 amino acids reduce cation-π binding energies but have van der Waals radii similar to that of phenylalanine. The activities of the SHC variants with fluorophenylalanines were found to be inversely proportional to the number of the fluorine atoms on the aromatic ring and clearly correlated with the cation-π binding energies of the ring moiety. No serious structural alteration was observed for these variants even at high temperature. These results unambiguously show that the π-electron density of residues 365 and 605 has a crucial role for the efficient polycyclization reaction by SHC. This is the first report to demonstrate experimentally the involvement of cation-π interaction in triterpene biosynthesis.

AB - It has been assumed that the π-electrons of aromatic residues in the catalytic sites of triterpene cyclases stabilize the cationic intermediates formed during the polycyclization cascade of squalene or oxidosqualene, but no definitive experimental evidence has been given. To validate this cation-π 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 of the wild-type SHC. The catalytic sites of Phe365 and Phe605 were substituted with O-methyltyrosine, tyrosine, and tryptophan, which have higher cation-π binding energies than phenylalanine. These replacements actually increased the SHC activity at low temperature, but decreased the activity at high temperature, as compared with the wild-type SHC. This decreased activity is due to the disorganization of the 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 amino acids reduce cation-π binding energies but have van der Waals radii similar to that of phenylalanine. The activities of the SHC variants with fluorophenylalanines were found to be inversely proportional to the number of the fluorine atoms on the aromatic ring and clearly correlated with the cation-π binding energies of the ring moiety. No serious structural alteration was observed for these variants even at high temperature. These results unambiguously show that the π-electron density of residues 365 and 605 has a crucial role for the efficient polycyclization reaction by SHC. This is the first report to demonstrate experimentally the involvement of cation-π interaction in triterpene biosynthesis.

UR - http://www.scopus.com/inward/record.url?scp=33749529751&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=33749529751&partnerID=8YFLogxK

U2 - 10.1021/ja063358p

DO - 10.1021/ja063358p

M3 - Article

VL - 128

SP - 13184

EP - 13194

JO - Journal of the American Chemical Society

JF - Journal of the American Chemical Society

SN - 0002-7863

IS - 40

ER -