The hyperthermophilic cystathionine γ-synthase from the aerobic crenarchaeon Sulfolobus tokodaii: expression, purification, crystallization and structural insights

Autor: Mai Shinozaki, Kenji Inagaki, Tetsuya Yamada, Shigeharu Harada, Sae Mizuno, Dan Sato, Shoko Hanada, Masahiko Yanagitani, Tomoo Shiba, Ayaka Kawamura, Takashi Tamura
Rok vydání: 2016
Předmět:
0301 basic medicine
Models
Molecular
Hot Temperature
Stereochemistry
Archaeal Proteins
Carbon-Oxygen Lyases
Biophysics
Sulfolobus tokodaii
Gene Expression
Transsulfuration
Transsulfuration pathway
Crystallography
X-Ray
Biochemistry
law.invention
Polyethylene Glycols
Substrate Specificity
Sulfolobus
Research Communications
03 medical and health sciences
chemistry.chemical_compound
Glycols
Structural Biology
law
Genetics
Escherichia coli
Chemical Precipitation
Amino Acid Sequence
Crystallization
Cloning
Molecular
Pyridoxal
chemistry.chemical_classification
Binding Sites
biology
Sequence Homology
Amino Acid
Thermophile
Hydrogen-Ion Concentration
Condensed Matter Physics
Cystathionine beta synthase
Recombinant Proteins
Kinetics
030104 developmental biology
Enzyme
chemistry
Pyridoxal Phosphate
biology.protein
Sequence Alignment
Plasmids
Protein Binding
Zdroj: Acta crystallographica. Section F, Structural biology communications. 73(Pt 3)
ISSN: 2053-230X
Popis: 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 archaeonSulfolobus tokodaii(StCGS) was overexpressed inEscherichia coliand 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 groupP21; unit-cell parametersa= 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 groupC2221; unit-cell parametersa= 117.7,b= 117.8,c= 251.3 Å) and in form II′ at 313 K (space groupC2221; unit-cell parametersa= 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.
Databáze: OpenAIRE
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