TY - JOUR
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.
PY - 2004/7/30
Y1 - 2004/7/30
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.
UR - http://www.scopus.com/inward/record.url?scp=3543014927&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=3543014927&partnerID=8YFLogxK
U2 - 10.1074/jbc.M404381200
DO - 10.1074/jbc.M404381200
M3 - Article
C2 - 15155761
AN - SCOPUS:3543014927
SN - 0021-9258
VL - 279
SP - 32418
EP - 32425
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
IS - 31
ER -