title: Escherichia coli L-serine deaminase requires a [4Fe-4S] cluster in catalysis.
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 axially = 2.03 and g radially = 1.93. Mössbauer spectra of the (57)Fe-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, delta = 0.47 mm/s, quadrupole splitting of Delta E(Q) = 1.14 mm/s, and a diamagnetic (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.
authors.affiliation: Department of Biochemistry and Molecular Biology, The Pennsylvania State University, University Park, 16802, USA.
authors.forename: Robert M
authors.initials: RM
authors.lastname: Cicchillo
authors.forename: Melissa A
authors.initials: MA
authors.lastname: Baker
authors.forename: Eric J
authors.initials: EJ
authors.lastname: Schnitzer
authors.forename: Elaine B
authors.initials: EB
authors.lastname: Newman
authors.forename: Carsten
authors.initials: C
authors.lastname: Krebs
authors.forename: Squire J
authors.initials: SJ
authors.lastname: Booker
mesh_terms: D001667:Binding, Competitive; D002384:Catalysis; D002845:Chromatography; D004227:Dithionite / Q000737:chemistry; D004305:Dose-Response Relationship, Drug; D004578:Electron Spin Resonance Spectroscopy; D004926:Escherichia coli / Q000201:enzymology*; D007501:Iron / Q000737:chemistry; D007506:Iron-Sulfur Proteins / Q000737:chemistry*; D007700:Kinetics; D012695:L-Serine Dehydratase / Q000737:chemistry*; D008280:Magnetics; D008956:Models, Chemical; D010455:Peptides / Q000737:chemistry; D012694:Serine / Q000737:chemistry; D015398:Signal Transduction; D013053:Spectrophotometry; D015204:Spectroscopy, Mossbauer; D013440:Sulfides / Q000737:chemistry; D013816:Thermodynamics; D013997:Time Factors; D014466:Ultraviolet Rays
chemical_list: D007506:Iron-Sulfur Proteins; D010455:Peptides; D013440:Sulfides; D004227:Dithionite; D012694:Serine; D007501:Iron; D012695:L-Serine Dehydratase