The hyperthermophilic cystathionine γ-synthase from the aerobic crenarchaeonSulfolobus 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 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.