RESUMEN

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, ß = 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 Šresolution 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.

Asunto(s)

Proteínas Arqueales/química , Liasas de Carbono-Oxígeno/química , Fosfato de Piridoxal/química , Sulfolobus/química , Secuencia de Aminoácidos , Proteínas Arqueales/genética , Proteínas Arqueales/metabolismo , Sitios de Unión , Liasas de Carbono-Oxígeno/genética , Liasas de Carbono-Oxígeno/metabolismo , Precipitación Química , Clonación Molecular , Cristalografía por Rayos X , Escherichia coli/genética , Escherichia coli/metabolismo , Expresión Génica , Glicoles/química , Calor , Concentración de Iones de Hidrógeno , Cinética , Modelos Moleculares , Plásmidos/química , Plásmidos/metabolismo , Polietilenglicoles/química , Unión Proteica , Fosfato de Piridoxal/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Especificidad por Sustrato , Sulfolobus/enzimología