title: Pyruvate formate-lyase-activating enzyme: strictly anaerobic isolation yields active enzyme containing a [3Fe-4S](+) cluster.
abstract: Pyruvate formate-lyase-activating enzyme (PFL-AE) from Escherichia coli (E. coli) catalyzes the stereospecific abstraction of a hydrogen atom from Gly734 of pyruvate formate-lyase (PFL) in a reaction that is strictly dependent on the cosubstrate S-adenosyl-l-methionine (AdoMet). Although PFL-AE is an iron-dependent enzyme, isolation of the enzyme with its metal center intact has proven difficult due to the oxygen sensitivity and lability of the metal center. We report here the first isolation of PFL-AE under nondenaturing, strictly anaerobic conditions. Iron and sulfide analysis as well as UV-visible, EPR, and resonance Raman data support the presence of a [3Fe-4S](+) cluster in the purified enzyme. The isolated native enzyme, but not apo-enzyme, exhibits a high specific activity (31 U/mg) in the absence of added iron, indicating that the native cluster is necessary and sufficient for enzymatic activity.
authors.affiliation: Department of Chemistry, Michigan State University, East Lansing, Michigan 48824, USA. broderj@cem.msu.edu
authors.forename: J B
authors.initials: JB
authors.lastname: Broderick
authors.forename: T F
authors.initials: TF
authors.lastname: Henshaw
authors.forename: J
authors.initials: J
authors.lastname: Cheek
authors.forename: K
authors.initials: K
authors.lastname: Wojtuszewski
authors.forename: S R
authors.initials: SR
authors.lastname: Smith
authors.forename: M R
authors.initials: MR
authors.lastname: Trojan
authors.forename: R M
authors.initials: RM
authors.lastname: McGhan
authors.forename: A
authors.initials: A
authors.lastname: Kopf
authors.forename: M
authors.initials: M
authors.lastname: Kibbey
authors.forename: W E
authors.initials: WE
authors.lastname: Broderick
mesh_terms: D000123:Acetyltransferases; D004578:Electron Spin Resonance Spectroscopy; D004798:Enzymes / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D007506:Iron-Sulfur Proteins / Q000032:analysis*
chemical_list: D004798:Enzymes; D007506:Iron-Sulfur Proteins; D000123:Acetyltransferases; C040285:pyruvate formate-lyase activating enzyme