Production and isolation of vanadium nitrogenase from Azotobacter vinelandii by molybdenum depletion

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• 作者：Daniel Sippel ; Julia Schlesier…
• 关键词：Biological nitrogen fixation ; Alternative nitrogenase ; Vanadium ; VFe protein ; Anoxic protein biochemistry
• 刊名：JBIC Journal of Biological Inorganic Chemistry
• 出版年：2017
• 出版时间：January 2017
• 年：2017
• 卷：22
• 期：1
• 页码：161-168
• 全文大小：
• 刊物类别：Biomedical and Life Sciences
• 刊物主题：Biochemistry, general; Microbiology;
• 出版者：Springer Berlin Heidelberg
• ISSN：1432-1327
• 卷排序：22

文摘

The alternative, vanadium-dependent nitrogenase is employed by Azotobacter vinelandii for the fixation of atmospheric N₂ under 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 VnfD₂K₂G₂ heterohexamer.