title: The hyperthermophilic cystathionine γ-synthase from the aerobic crenarchaeon Sulfolobus tokodaii: expression, purification, crystallization and structural insights.
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.
authors.affiliation: Department of Applied Biology, Kyoto Institute of Technology, Gosho Kaido-cho, Matsugasaki, Sakyo-ku, Kyoto 606-8585, Japan.
authors.forename: Dan
authors.initials: D
authors.lastname: Sato
authors.affiliation: Department of Applied Biology, Kyoto Institute of Technology, Gosho Kaido-cho, Matsugasaki, Sakyo-ku, Kyoto 606-8585, Japan.
authors.forename: Tomoo
authors.initials: T
authors.lastname: Shiba
authors.affiliation: Department of Applied Biology, Kyoto Institute of Technology, Gosho Kaido-cho, Matsugasaki, Sakyo-ku, Kyoto 606-8585, Japan.
authors.forename: Sae
authors.initials: S
authors.lastname: Mizuno
authors.affiliation: Department of Applied Biology, Kyoto Institute of Technology, Gosho Kaido-cho, Matsugasaki, Sakyo-ku, Kyoto 606-8585, Japan.
authors.forename: Ayaka
authors.initials: A
authors.lastname: Kawamura
authors.affiliation: Department of Applied Biology, Kyoto Institute of Technology, Gosho Kaido-cho, Matsugasaki, Sakyo-ku, Kyoto 606-8585, Japan.
authors.forename: Shoko
authors.initials: S
authors.lastname: Hanada
authors.affiliation: Department of Biofunctional Chemistry, Okayama University, Tsushima-naka 1-1-1, Kita-ku, Okayama 700-8530, Japan.
authors.forename: Tetsuya
authors.initials: T
authors.lastname: Yamada
authors.affiliation: Department of Biofunctional Chemistry, Okayama University, Tsushima-naka 1-1-1, Kita-ku, Okayama 700-8530, Japan.
authors.forename: Mai
authors.initials: M
authors.lastname: Shinozaki
authors.affiliation: Department of Biofunctional Chemistry, Okayama University, Tsushima-naka 1-1-1, Kita-ku, Okayama 700-8530, Japan.
authors.forename: Masahiko
authors.initials: M
authors.lastname: Yanagitani
authors.affiliation: Department of Biofunctional Chemistry, Okayama University, Tsushima-naka 1-1-1, Kita-ku, Okayama 700-8530, Japan.
authors.forename: Takashi
authors.initials: T
authors.lastname: Tamura
authors.affiliation: Department of Biofunctional Chemistry, Okayama University, Tsushima-naka 1-1-1, Kita-ku, Okayama 700-8530, Japan.
authors.forename: Kenji
authors.initials: K
authors.lastname: Inagaki
authors.affiliation: Department of Applied Biology, Kyoto Institute of Technology, Gosho Kaido-cho, Matsugasaki, Sakyo-ku, Kyoto 606-8585, Japan.
authors.forename: Shigeharu
authors.initials: S
authors.lastname: Harada
mesh_terms: D000595:Amino Acid Sequence; D019843:Archaeal Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D001665:Binding Sites; D019757:Carbon-Oxygen Lyases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011232:Chemical Precipitation; D003001:Cloning, Molecular; D018360:Crystallography, X-Ray; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D006018:Glycols / Q000737:chemistry; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008958:Models, Molecular; D010957:Plasmids / Q000737:chemistry / Q000378:metabolism; D011092:Polyethylene Glycols / Q000737:chemistry; D011485:Protein Binding; D011732:Pyridoxal Phosphate / Q000737:chemistry* / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D016944:Sulfolobus / Q000737:chemistry* / Q000201:enzymology
chemical_list: D019843:Archaeal Proteins; D006018:Glycols; D011994:Recombinant Proteins; D011092:Polyethylene Glycols; D011732:Pyridoxal Phosphate; C022696:O-succinylhomoserine (thiol)-lyase; D019757:Carbon-Oxygen Lyases; C000595212:polyethylene glycol 3350; C011480:hexylene glycol
keywords: Sulfolobus tokodaii; cystathionine γ-synthase; hyperthermophilic enzyme; methionine biosynthesis; pyridoxal 5′-phosphate; transsulfuration