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

doi:10.1016/j.biochi.2004.06.003

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

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11 Scopus citations

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 language	English
Pages (from-to)	481-485
Number of pages	5
Journal	Biochimie
Volume	86
Issue number	7
DOIs	https://doi.org/10.1016/j.biochi.2004.06.003
State	Published - Jul 2004
Externally published	Yes

Bibliographical note

Funding 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.

Keywords

alpha;-Aminoacrylate, Geobacillus stearothermophilus
Cysteine biosynthesis
Cysteine synthase

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10.1016/j.biochi.2004.06.003

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@article{a23fcf1c6a3a423cae354a1382093aae,

title = "Biochemical characterization of a thermostable cysteine synthase from Geobacillus stearothermophilus V",

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.",

keywords = "alpha;-Aminoacrylate, Geobacillus stearothermophilus, Cysteine biosynthesis, Cysteine synthase",

author = "Saavedra, {C. P.} and Encinas, {M. V.} and Araya, {M. A.} and P{\'e}rez, {J. M.} and Tantale{\'a}n, {J. C.} and Fuentes, {D. E.} and Calder{\'o}n, {I. L.} and Pichuantes, {S. E.} and V{\'a}squez, {C. C.}",

note = "Funding 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.",

year = "2004",

month = jul,

doi = "10.1016/j.biochi.2004.06.003",

language = "Ingl{\'e}s",

volume = "86",

pages = "481--485",

journal = "Biochimie",

issn = "0300-9084",

publisher = "Elsevier",

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TY - JOUR

T1 - Biochemical characterization of a thermostable cysteine synthase from Geobacillus stearothermophilus V

AU - Saavedra, C. P.

AU - Encinas, M. V.

AU - Araya, M. A.

AU - Pérez, J. M.

AU - Tantaleán, J. C.

AU - Fuentes, D. E.

AU - Calderón, I. L.

AU - Pichuantes, S. E.

AU - Vásquez, C. C.

N1 - Funding 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.

PY - 2004/7

Y1 - 2004/7

N2 - 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.

AB - 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.

KW - alpha;-Aminoacrylate, Geobacillus stearothermophilus

KW - Cysteine biosynthesis

KW - Cysteine synthase

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U2 - 10.1016/j.biochi.2004.06.003

DO - 10.1016/j.biochi.2004.06.003

M3 - Artículo

C2 - 15308337

AN - SCOPUS:4143061457

SN - 0300-9084

VL - 86

SP - 481

EP - 485

JO - Biochimie

JF - Biochimie

IS - 7

ER -

Biochemical characterization of a thermostable cysteine synthase from Geobacillus stearothermophilus V

Abstract

Bibliographical note

Keywords

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