The hyperthermophilic cystathionine c-synthase from the aerobic crenarchaeon Sulfolobus tokodaii

Expression, purification, crystallization and structural insights

Dan Sato, Tomoo Shiba, Sae Mizuno, Ayaka Kawamura, Shoko Hanada, Tetsuya Yamada, Mai Shinozaki, Masahiko Yanagitani, Takashi Tamura, Kenji Inagaki, Shigeharu Harada

Research output: Contribution to journalArticle

Abstract

Cystathionine γ-synthase (CGS; EC 2.5.1.48), a pyridoxal 5′-phosphate (PLP)-dependent enzyme, catalyzes the formation of cystathionine from an l-homoserine derivative and l-cysteine in the first step of the transsulfuration pathway. Recombinant CGS from the thermoacidophilic archaeon Sulfolobus tokodaii (StCGS) was overexpressed in Escherichia coli and purified to homogeneity by heat treatment followed by hydroxyapatite and gel-filtration column chromatography. The purified enzyme shows higher enzymatic activity at 353 K under basic pH conditions compared with that at 293 K. Crystallization trials yielded three crystal forms from different temperature and pH conditions. Form I crystals (space group P21; unit-cell parameters a = 58.4, b = 149.3, c = 90.2 Å, β = 108.9°) were obtained at 293 K under acidic pH conditions using 2-methyl-2,4-pentanediol as a precipitant, whereas under basic pH conditions the enzyme crystallized in form II at 293 K (space group C2221; unit-cell parameters a = 117.7, b = 117.8, c = 251.3 Å) and in form II′ at 313 K (space group C2221; unit-cell parameters a = 107.5, b = 127.7, c = 251.1 Å) using polyethylene glycol 3350 as a precipitant. X-ray diffraction data were collected to 2.2, 2.9 and 2.7 Å resolution for forms I, II and II′, respectively. Structural analysis of these crystal forms shows that the orientation of the bound PLP in form II is significantly different from that in form II′, suggesting that the change in orientation of PLP with temperature plays a role in the thermophilic enzymatic activity of StCGS.

Original languageEnglish
Pages (from-to)152-158
Number of pages7
JournalActa Crystallographica Section:F Structural Biology Communications
Volume73
DOIs
Publication statusPublished - 2017

Fingerprint

Sulfolobus
Cystathionine
Pyridoxal Phosphate
Crystallization
purification
Purification
crystallization
enzymes
phosphates
Crystals
Enzymes
Homoserine
Column chromatography
cells
Durapatite
crystals
Structural analysis
Crystal orientation
Escherichia coli
Temperature

Keywords

  • Cystathionineγ-synthase.
  • Hyperthermophilic enzyme
  • Methionine biosynthesis
  • Pyridoxal 50-phosphate
  • Sulfolobus tokodaii
  • Transsulfuration

ASJC Scopus subject areas

  • Structural Biology
  • Biophysics
  • Biochemistry
  • Genetics
  • Condensed Matter Physics

Cite this

The hyperthermophilic cystathionine c-synthase from the aerobic crenarchaeon Sulfolobus tokodaii : Expression, purification, crystallization and structural insights. / Sato, Dan; Shiba, Tomoo; Mizuno, Sae; Kawamura, Ayaka; Hanada, Shoko; Yamada, Tetsuya; Shinozaki, Mai; Yanagitani, Masahiko; Tamura, Takashi; Inagaki, Kenji; Harada, Shigeharu.

In: Acta Crystallographica Section:F Structural Biology Communications, Vol. 73, 2017, p. 152-158.

Research output: Contribution to journalArticle

Sato, Dan ; Shiba, Tomoo ; Mizuno, Sae ; Kawamura, Ayaka ; Hanada, Shoko ; Yamada, Tetsuya ; Shinozaki, Mai ; Yanagitani, Masahiko ; Tamura, Takashi ; Inagaki, Kenji ; Harada, Shigeharu. / The hyperthermophilic cystathionine c-synthase from the aerobic crenarchaeon Sulfolobus tokodaii : Expression, purification, crystallization and structural insights. In: Acta Crystallographica Section:F Structural Biology Communications. 2017 ; Vol. 73. pp. 152-158.
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abstract = "Cystathionine γ-synthase (CGS; EC 2.5.1.48), a pyridoxal 5′-phosphate (PLP)-dependent enzyme, catalyzes the formation of cystathionine from an l-homoserine derivative and l-cysteine in the first step of the transsulfuration pathway. Recombinant CGS from the thermoacidophilic archaeon Sulfolobus tokodaii (StCGS) was overexpressed in Escherichia coli and purified to homogeneity by heat treatment followed by hydroxyapatite and gel-filtration column chromatography. The purified enzyme shows higher enzymatic activity at 353 K under basic pH conditions compared with that at 293 K. Crystallization trials yielded three crystal forms from different temperature and pH conditions. Form I crystals (space group P21; unit-cell parameters a = 58.4, b = 149.3, c = 90.2 {\AA}, β = 108.9°) were obtained at 293 K under acidic pH conditions using 2-methyl-2,4-pentanediol as a precipitant, whereas under basic pH conditions the enzyme crystallized in form II at 293 K (space group C2221; unit-cell parameters a = 117.7, b = 117.8, c = 251.3 {\AA}) and in form II′ at 313 K (space group C2221; unit-cell parameters a = 107.5, b = 127.7, c = 251.1 {\AA}) using polyethylene glycol 3350 as a precipitant. X-ray diffraction data were collected to 2.2, 2.9 and 2.7 {\AA} resolution for forms I, II and II′, respectively. Structural analysis of these crystal forms shows that the orientation of the bound PLP in form II is significantly different from that in form II′, suggesting that the change in orientation of PLP with temperature plays a role in the thermophilic enzymatic activity of StCGS.",
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author = "Dan Sato and Tomoo Shiba and Sae Mizuno and Ayaka Kawamura and Shoko Hanada and Tetsuya Yamada and Mai Shinozaki and Masahiko Yanagitani and Takashi Tamura and Kenji Inagaki and Shigeharu Harada",
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T1 - The hyperthermophilic cystathionine c-synthase from the aerobic crenarchaeon Sulfolobus tokodaii

T2 - Expression, purification, crystallization and structural insights

AU - Sato, Dan

AU - Shiba, Tomoo

AU - Mizuno, Sae

AU - Kawamura, Ayaka

AU - Hanada, Shoko

AU - Yamada, Tetsuya

AU - Shinozaki, Mai

AU - Yanagitani, Masahiko

AU - Tamura, Takashi

AU - Inagaki, Kenji

AU - Harada, Shigeharu

PY - 2017

Y1 - 2017

N2 - Cystathionine γ-synthase (CGS; EC 2.5.1.48), a pyridoxal 5′-phosphate (PLP)-dependent enzyme, catalyzes the formation of cystathionine from an l-homoserine derivative and l-cysteine in the first step of the transsulfuration pathway. Recombinant CGS from the thermoacidophilic archaeon Sulfolobus tokodaii (StCGS) was overexpressed in Escherichia coli and purified to homogeneity by heat treatment followed by hydroxyapatite and gel-filtration column chromatography. The purified enzyme shows higher enzymatic activity at 353 K under basic pH conditions compared with that at 293 K. Crystallization trials yielded three crystal forms from different temperature and pH conditions. Form I crystals (space group P21; unit-cell parameters a = 58.4, b = 149.3, c = 90.2 Å, β = 108.9°) were obtained at 293 K under acidic pH conditions using 2-methyl-2,4-pentanediol as a precipitant, whereas under basic pH conditions the enzyme crystallized in form II at 293 K (space group C2221; unit-cell parameters a = 117.7, b = 117.8, c = 251.3 Å) and in form II′ at 313 K (space group C2221; unit-cell parameters a = 107.5, b = 127.7, c = 251.1 Å) using polyethylene glycol 3350 as a precipitant. X-ray diffraction data were collected to 2.2, 2.9 and 2.7 Å resolution for forms I, II and II′, respectively. Structural analysis of these crystal forms shows that the orientation of the bound PLP in form II is significantly different from that in form II′, suggesting that the change in orientation of PLP with temperature plays a role in the thermophilic enzymatic activity of StCGS.

AB - Cystathionine γ-synthase (CGS; EC 2.5.1.48), a pyridoxal 5′-phosphate (PLP)-dependent enzyme, catalyzes the formation of cystathionine from an l-homoserine derivative and l-cysteine in the first step of the transsulfuration pathway. Recombinant CGS from the thermoacidophilic archaeon Sulfolobus tokodaii (StCGS) was overexpressed in Escherichia coli and purified to homogeneity by heat treatment followed by hydroxyapatite and gel-filtration column chromatography. The purified enzyme shows higher enzymatic activity at 353 K under basic pH conditions compared with that at 293 K. Crystallization trials yielded three crystal forms from different temperature and pH conditions. Form I crystals (space group P21; unit-cell parameters a = 58.4, b = 149.3, c = 90.2 Å, β = 108.9°) were obtained at 293 K under acidic pH conditions using 2-methyl-2,4-pentanediol as a precipitant, whereas under basic pH conditions the enzyme crystallized in form II at 293 K (space group C2221; unit-cell parameters a = 117.7, b = 117.8, c = 251.3 Å) and in form II′ at 313 K (space group C2221; unit-cell parameters a = 107.5, b = 127.7, c = 251.1 Å) using polyethylene glycol 3350 as a precipitant. X-ray diffraction data were collected to 2.2, 2.9 and 2.7 Å resolution for forms I, II and II′, respectively. Structural analysis of these crystal forms shows that the orientation of the bound PLP in form II is significantly different from that in form II′, suggesting that the change in orientation of PLP with temperature plays a role in the thermophilic enzymatic activity of StCGS.

KW - Cystathionineγ-synthase.

KW - Hyperthermophilic enzyme

KW - Methionine biosynthesis

KW - Pyridoxal 50-phosphate

KW - Sulfolobus tokodaii

KW - Transsulfuration

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