The cysK gene encoding a cysteine synthase of Geobacillus stearothermophilus V was overexpressed in E. coli and the recombinant protein was purified and characterized. The enzyme is a thermostable homodimer (32 kDa/monomer) belonging to the β family of pyridoxal phosphate (PLP)-dependent enzymes. UV-visible spectra showed absorption bands at 279 and 410 nm. The band at 279 nm is due to tyrosine residues as the enzyme lacks tryptophan. The 410 nm band represents absorption of the coenzyme bound as a Schiff base to a lysine residue of the protein. Fluorescence characteristics of CysK's Schiff base were influenced by temperature changes suggesting different local structures at the cofactor binding site. The emission of the Schiff base allowed the determination of binding constants for products at both 20°C and 50°C. At 50°C and in the absence of sulphide the enzyme catalyzes the decomposition of O-acetyl-l-serine to pyruvate and ammonia. At 20°C, however, a stable α-aminoacrylate intermediate is formed.
Bibliographical noteFunding Information:
The authors are indebted to Dr. Emilio Cardemil for critical reading of the manuscript. J.C.T. was supported by a doctoral fellowship of the DAAD (Germany). M.A.A., J.M.P., D.E.F. and I.L.C. were supported by doctoral fellowships from MECESUP (Chile). This work was supported in part by grants 1990917 and 1030234 from Fondecyt and Dicyt.
- alpha;-Aminoacrylate, Geobacillus stearothermophilus
- Cysteine biosynthesis
- Cysteine synthase