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Biochemical characterization of a thermostable cysteine synthase from Geobacillus stearothermophilus V. 2004

C P Saavedra, and M V Encinas, and M A Araya, and J M Pérez, and J C Tantaleán, and D E Fuentes, and I L Calderón, and S E Pichuantes, and C C Vásquez
Laboratorio de Microbiología Molecular, Facultad de Química y Biología, Universidad de Santiago de Chile, Casilla 40, Correo 33, Santiago, Chile.

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 beta 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 degrees C and 50 degrees C. At 50 degrees C and in the absence of sulphide the enzyme catalyzes the decomposition of O-acetyl-l-serine to pyruvate and ammonia. At 20 degrees C, however, a stable alpha-aminoacrylate intermediate is formed.

UI MeSH Term Description Entries
D007700 Kinetics The rate dynamics in chemical or physical systems.
D003547 Cysteine Synthase An enzyme that catalyzes the biosynthesis of cysteine in microorganisms and plants from O-acetyl-L-serine and hydrogen sulfide. This enzyme was formerly listed as EC 4.2.99.8. O-Acetylserine Sulfhydrylase,Cysteine Synthase A,O-Acetylserine (Thiol)-Lyase,O Acetylserine Sulfhydrylase,Sulfhydrylase, O-Acetylserine
D004795 Enzyme Stability The extent to which an enzyme retains its structural conformation or its activity when subjected to storage, isolation, and purification or various other physical or chemical manipulations, including proteolytic enzymes and heat. Enzyme Stabilities,Stabilities, Enzyme,Stability, Enzyme
D001406 Bacillaceae A family of bacteria which produce endospores. They are mostly saprophytes from soil, but a few are insect or animal parasites or pathogens.
D013050 Spectrometry, Fluorescence Measurement of the intensity and quality of fluorescence. Fluorescence Spectrophotometry,Fluorescence Spectroscopy,Spectrofluorometry,Fluorescence Spectrometry,Spectrophotometry, Fluorescence,Spectroscopy, Fluorescence
D013053 Spectrophotometry The art or process of comparing photometrically the relative intensities of the light in different parts of the spectrum.
D013816 Thermodynamics A rigorously mathematical analysis of energy relationships (heat, work, temperature, and equilibrium). It describes systems whose states are determined by thermal parameters, such as temperature, in addition to mechanical and electromagnetic parameters. (From Hawley's Condensed Chemical Dictionary, 12th ed) Thermodynamic

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