title: Production and isolation of vanadium nitrogenase from Azotobacter vinelandii by molybdenum depletion.
abstract: The alternative, vanadium-dependent nitrogenase is employed by Azotobacter vinelandii for the fixation of atmospheric N2under conditions of molybdenum starvation. While overall similar in architecture and functionality to the common Mo-nitrogenase, the V-dependent enzyme exhibits a series of unique features that on one hand are of high interest for biotechnological applications. As its catalytic properties differ from Mo-nitrogenase, it may on the other hand also provide invaluable clues regarding the molecular mechanism of biological nitrogen fixation that remains scarcely understood to date. Earlier studies on vanadium nitrogenase were almost exclusively based on a ΔnifHDK strain of A. vinelandii, later also in a version with a hexahistidine affinity tag on the enzyme. As structural analyses remained unsuccessful with such preparations we have developed protocols to isolate unmodified vanadium nitrogenase from molybdenum-depleted, actively nitrogen-fixing A. vinelandii wild-type cells. The procedure provides pure protein at high yields whose spectroscopic properties strongly resemble data presented earlier. Analytical size-exclusion chromatography shows this preparation to be a VnfD2K2G2heterohexamer.
authors.affiliation: Institute for Biochemistry, Albert-Ludwigs-University Freiburg, Albertstraße 21, 79104, Freiburg Im Breisgau, Germany.|BIOSS Centre for Biological Signalling Studies, 79104, Freiburg, Germany.
authors.forename: Daniel
authors.initials: D
authors.lastname: Sippel
authors.affiliation: Institute for Biochemistry, Albert-Ludwigs-University Freiburg, Albertstraße 21, 79104, Freiburg Im Breisgau, Germany.|BIOSS Centre for Biological Signalling Studies, 79104, Freiburg, Germany.
authors.forename: Julia
authors.initials: J
authors.lastname: Schlesier
authors.affiliation: Institute for Biochemistry, Albert-Ludwigs-University Freiburg, Albertstraße 21, 79104, Freiburg Im Breisgau, Germany.|BIOSS Centre for Biological Signalling Studies, 79104, Freiburg, Germany.
authors.forename: Michael
authors.initials: M
authors.lastname: Rohde
authors.affiliation: Institute for Biochemistry, Albert-Ludwigs-University Freiburg, Albertstraße 21, 79104, Freiburg Im Breisgau, Germany.|BIOSS Centre for Biological Signalling Studies, 79104, Freiburg, Germany.
authors.forename: Christian
authors.initials: C
authors.lastname: Trncik
authors.affiliation: Institute for Biochemistry, Albert-Ludwigs-University Freiburg, Albertstraße 21, 79104, Freiburg Im Breisgau, Germany.|BIOSS Centre for Biological Signalling Studies, 79104, Freiburg, Germany.
authors.forename: Laure
authors.initials: L
authors.lastname: Decamps
authors.affiliation: Institute for Biochemistry, Albert-Ludwigs-University Freiburg, Albertstraße 21, 79104, Freiburg Im Breisgau, Germany.|BIOSS Centre for Biological Signalling Studies, 79104, Freiburg, Germany.
authors.forename: Ivana
authors.initials: I
authors.lastname: Djurdjevic
authors.affiliation: Institute for Biochemistry, Albert-Ludwigs-University Freiburg, Albertstraße 21, 79104, Freiburg Im Breisgau, Germany.|BIOSS Centre for Biological Signalling Studies, 79104, Freiburg, Germany.
authors.forename: Thomas
authors.initials: T
authors.lastname: Spatzal
authors.affiliation: Institute for Biochemistry, Albert-Ludwigs-University Freiburg, Albertstraße 21, 79104, Freiburg Im Breisgau, Germany.|BIOSS Centre for Biological Signalling Studies, 79104, Freiburg, Germany.
authors.forename: Susana L A
authors.initials: SL
authors.lastname: Andrade
authors.affiliation: Institute for Biochemistry, Albert-Ludwigs-University Freiburg, Albertstraße 21, 79104, Freiburg Im Breisgau, Germany. einsle@biochemie.uni-freiburg.de.|BIOSS Centre for Biological Signalling Studies, 79104, Freiburg, Germany. einsle@biochemie.uni-freiburg.de.
authors.forename: Oliver
authors.initials: O
authors.lastname: Einsle
mesh_terms: D016948:Azotobacter vinelandii / Q000187:drug effects / Q000201:enzymology* / Q000254:growth & development / Q000378:metabolism; D055162:Biocatalysis; D003470:Culture Media / Q000737:chemistry; D004305:Dose-Response Relationship, Drug; D008982:Molybdenum / Q000494:pharmacology*; D009591:Nitrogenase / Q000096:biosynthesis* / Q000302:isolation & purification* / Q000378:metabolism
chemical_list: D003470:Culture Media; D008982:Molybdenum; D009591:Nitrogenase
keywords: Alternative nitrogenase; Anoxic protein biochemistry; Biological nitrogen fixation; VFe protein; Vanadium