Escherichia coli L-serine deaminase requires a [4Fe-4S] cluster in catalysis

Robert M. Cicchillo, Melissa A. Baker, Eric J. Schnitzer, Elaine B. Newman, Carsten Krebs, Squire J. Booker

Research output: Contribution to journalArticle

31 Citations (Scopus)

Abstract

L-Serine deaminases catalyze the deamination of L-serine, producing pyruvate and ammonia. Two families of these proteins have been described and are delineated by the cofactor that each employs in catalysis. These are the pyridoxal 5′-phosphate-dependent deaminases and the deaminases that are activated in vitro by iron and dithiothreitol. In contrast to the enzymes that employ pyridoxal 5′-phosphate, detailed physical and mechanistic characterization of the iron-dependent deaminases is limited, primarily because of their extreme instability. We report here the characterization of L-serine deaminase from Escherichia coli, which is the product of the sdaA gene. When purified anaerobically, the isolated protein contains 1.86 ± 0.46 eq of iron and 0.670 ± 0.019 eq of sulfide per polypeptide and displays a UV-visible spectrum that is consistent with a [4Fe-4S] cluster. Reconstitution of the protein with iron and sulfide generates considerably more of the cluster, and treatment of the reconstituted protein with dithionite gives rise to an axial EPR spectrum, displaying g∥ = 2.03 and g = 1.93. Mössbauer spectra of the 57Fe-reconstituted protein reveal that the majority of the iron is in the form of [4Fe-4S]2+ clusters, as evidenced by the typical Mössbauer parameters-isomer shift, δ = 0.47 mm/s, quadrupole splitting of ΔEQ = 1.14 mm/s, and a diamagmetic (S = 0) ground state. Treatment of the dithionite-reduced protein with L-serine results in a slight broadening of the feature at g = 2.03 in the EPR spectrum of the protein, and a dramatic loss in signal intensity, suggesting that the amino acid interacts directly with the cluster.

Original languageEnglish (US)
Pages (from-to)32418-32425
Number of pages8
JournalJournal of Biological Chemistry
Volume279
Issue number31
DOIs
StatePublished - Jul 30 2004

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L-Serine Dehydratase
Catalysis
Serine
Escherichia coli
Iron
Proteins
Dithionite
Pyridoxal Phosphate
Sulfides
Paramagnetic resonance
Deamination
Dithiothreitol
Pyruvic Acid
Ammonia
Isomers
Ground state
Genes
Amino Acids
Peptides

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

Cicchillo, Robert M. ; Baker, Melissa A. ; Schnitzer, Eric J. ; Newman, Elaine B. ; Krebs, Carsten ; Booker, Squire J. / Escherichia coli L-serine deaminase requires a [4Fe-4S] cluster in catalysis. In: Journal of Biological Chemistry. 2004 ; Vol. 279, No. 31. pp. 32418-32425.
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abstract = "L-Serine deaminases catalyze the deamination of L-serine, producing pyruvate and ammonia. Two families of these proteins have been described and are delineated by the cofactor that each employs in catalysis. These are the pyridoxal 5′-phosphate-dependent deaminases and the deaminases that are activated in vitro by iron and dithiothreitol. In contrast to the enzymes that employ pyridoxal 5′-phosphate, detailed physical and mechanistic characterization of the iron-dependent deaminases is limited, primarily because of their extreme instability. We report here the characterization of L-serine deaminase from Escherichia coli, which is the product of the sdaA gene. When purified anaerobically, the isolated protein contains 1.86 ± 0.46 eq of iron and 0.670 ± 0.019 eq of sulfide per polypeptide and displays a UV-visible spectrum that is consistent with a [4Fe-4S] cluster. Reconstitution of the protein with iron and sulfide generates considerably more of the cluster, and treatment of the reconstituted protein with dithionite gives rise to an axial EPR spectrum, displaying g∥ = 2.03 and g⊥ = 1.93. M{\"o}ssbauer spectra of the 57Fe-reconstituted protein reveal that the majority of the iron is in the form of [4Fe-4S]2+ clusters, as evidenced by the typical M{\"o}ssbauer parameters-isomer shift, δ = 0.47 mm/s, quadrupole splitting of ΔEQ = 1.14 mm/s, and a diamagmetic (S = 0) ground state. Treatment of the dithionite-reduced protein with L-serine results in a slight broadening of the feature at g = 2.03 in the EPR spectrum of the protein, and a dramatic loss in signal intensity, suggesting that the amino acid interacts directly with the cluster.",
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Escherichia coli L-serine deaminase requires a [4Fe-4S] cluster in catalysis. / Cicchillo, Robert M.; Baker, Melissa A.; Schnitzer, Eric J.; Newman, Elaine B.; Krebs, Carsten; Booker, Squire J.

In: Journal of Biological Chemistry, Vol. 279, No. 31, 30.07.2004, p. 32418-32425.

Research output: Contribution to journalArticle

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T1 - Escherichia coli L-serine deaminase requires a [4Fe-4S] cluster in catalysis

AU - Cicchillo, Robert M.

AU - Baker, Melissa A.

AU - Schnitzer, Eric J.

AU - Newman, Elaine B.

AU - Krebs, Carsten

AU - Booker, Squire J.

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N2 - L-Serine deaminases catalyze the deamination of L-serine, producing pyruvate and ammonia. Two families of these proteins have been described and are delineated by the cofactor that each employs in catalysis. These are the pyridoxal 5′-phosphate-dependent deaminases and the deaminases that are activated in vitro by iron and dithiothreitol. In contrast to the enzymes that employ pyridoxal 5′-phosphate, detailed physical and mechanistic characterization of the iron-dependent deaminases is limited, primarily because of their extreme instability. We report here the characterization of L-serine deaminase from Escherichia coli, which is the product of the sdaA gene. When purified anaerobically, the isolated protein contains 1.86 ± 0.46 eq of iron and 0.670 ± 0.019 eq of sulfide per polypeptide and displays a UV-visible spectrum that is consistent with a [4Fe-4S] cluster. Reconstitution of the protein with iron and sulfide generates considerably more of the cluster, and treatment of the reconstituted protein with dithionite gives rise to an axial EPR spectrum, displaying g∥ = 2.03 and g⊥ = 1.93. Mössbauer spectra of the 57Fe-reconstituted protein reveal that the majority of the iron is in the form of [4Fe-4S]2+ clusters, as evidenced by the typical Mössbauer parameters-isomer shift, δ = 0.47 mm/s, quadrupole splitting of ΔEQ = 1.14 mm/s, and a diamagmetic (S = 0) ground state. Treatment of the dithionite-reduced protein with L-serine results in a slight broadening of the feature at g = 2.03 in the EPR spectrum of the protein, and a dramatic loss in signal intensity, suggesting that the amino acid interacts directly with the cluster.

AB - L-Serine deaminases catalyze the deamination of L-serine, producing pyruvate and ammonia. Two families of these proteins have been described and are delineated by the cofactor that each employs in catalysis. These are the pyridoxal 5′-phosphate-dependent deaminases and the deaminases that are activated in vitro by iron and dithiothreitol. In contrast to the enzymes that employ pyridoxal 5′-phosphate, detailed physical and mechanistic characterization of the iron-dependent deaminases is limited, primarily because of their extreme instability. We report here the characterization of L-serine deaminase from Escherichia coli, which is the product of the sdaA gene. When purified anaerobically, the isolated protein contains 1.86 ± 0.46 eq of iron and 0.670 ± 0.019 eq of sulfide per polypeptide and displays a UV-visible spectrum that is consistent with a [4Fe-4S] cluster. Reconstitution of the protein with iron and sulfide generates considerably more of the cluster, and treatment of the reconstituted protein with dithionite gives rise to an axial EPR spectrum, displaying g∥ = 2.03 and g⊥ = 1.93. Mössbauer spectra of the 57Fe-reconstituted protein reveal that the majority of the iron is in the form of [4Fe-4S]2+ clusters, as evidenced by the typical Mössbauer parameters-isomer shift, δ = 0.47 mm/s, quadrupole splitting of ΔEQ = 1.14 mm/s, and a diamagmetic (S = 0) ground state. Treatment of the dithionite-reduced protein with L-serine results in a slight broadening of the feature at g = 2.03 in the EPR spectrum of the protein, and a dramatic loss in signal intensity, suggesting that the amino acid interacts directly with the cluster.

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