Biochemical characterization of a thermostable cysteine synthase from Geobacillus stearothermophilus V

C. P. Saavedra, M. V. Encinas, M. A. Araya, J. M. Pérez, J. C. Tantaleán, D. E. Fuentes, I. L. Calderón, S. E. Pichuantes, C. C. Vásquez

Research output: Contribution to journalArticle

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Abstract

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.

Original languageEnglish
Pages (from-to)481-485
Number of pages5
JournalBiochimie
Volume86
Issue number7
DOIs
Publication statusPublished - 1 Jul 2004

Fingerprint

Cysteine Synthase
Geobacillus stearothermophilus
Schiff Bases
Enzymes
Absorption spectra
Gene encoding
Pyridoxal Phosphate
Escherichia coli Proteins
Coenzymes
Sulfides
Pyruvic Acid
Ammonia
Recombinant Proteins
Tryptophan
Serine
Escherichia coli
Lysine
Tyrosine
Monomers
Fluorescence

Keywords

  • alpha;-Aminoacrylate, Geobacillus stearothermophilus
  • Cysteine biosynthesis
  • Cysteine synthase

ASJC Scopus subject areas

  • Biochemistry

Cite this

Saavedra, C. P. ; Encinas, M. V. ; Araya, M. A. ; Pérez, J. M. ; Tantaleán, J. C. ; Fuentes, D. E. ; Calderón, I. L. ; Pichuantes, S. E. ; Vásquez, C. C. / Biochemical characterization of a thermostable cysteine synthase from Geobacillus stearothermophilus V. In: Biochimie. 2004 ; Vol. 86, No. 7. pp. 481-485.
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Biochemical characterization of a thermostable cysteine synthase from Geobacillus stearothermophilus V. / Saavedra, C. P.; Encinas, M. V.; Araya, M. A.; Pérez, J. M.; Tantaleán, J. C.; Fuentes, D. E.; Calderón, I. L.; Pichuantes, S. E.; Vásquez, C. C.

In: Biochimie, Vol. 86, No. 7, 01.07.2004, p. 481-485.

Research output: Contribution to journalArticle

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AU - Pérez, J. M.

AU - Tantaleán, J. C.

AU - Fuentes, D. E.

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AU - Pichuantes, S. E.

AU - Vásquez, C. C.

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