Rettberg Lee, Tanifuji Kazuki, Jasniewski Andrew, Ribbe Markus Walter, Hu Yilin
Department of Molecular Biology and Biochemistry, University of California, Irvine, CA, United States.
Department of Molecular Biology and Biochemistry, University of California, Irvine, CA, United States; Department of Chemistry, University of California, Irvine, CA, United States.
Methods Enzymol. 2018;606:341-361. doi: 10.1016/bs.mie.2018.04.009. Epub 2018 May 10.
Nitrogenase is the only known enzymatic system that converts atmospheric dinitrogen (N) into bioavailable ammonia (NH). The active-site cofactor responsible for this reactivity is a [(R-homocitrate)MoFeSC] cluster that is designated as the M-cluster. This important cofactor is assembled stepwise from a pair of [FeS] clusters that become fused into a [FeSC] core before additional refinements take place to complete the biosynthesis. NifB, a member of the radical S-adenosyl-l-methionine (SAM) superfamily, facilitates the conversion of the [FeS] clusters (called the K-cluster) to the [FeSC] core (called the L-cluster). This transformation includes a SAM-dependent carbide insertion with concomitant incorporation of an additional sulfur. While difficulties with the purification of NifB have historically prevented detailed biochemical analyses, we have developed a heterologous expression system in Escherichia coli that yields stable NifB proteins from various N-fixing methanogenic organisms that can be used for studies. This chapter details the procedures necessary to prepare an active NifB protein. The methods used for the biochemical characterization of the SAM-dependent carbide insertion reactions are also described.
固氮酶是唯一已知的能将大气中的双氮（N₂）转化为生物可利用氨（NH₃）的酶系统。负责这种反应活性的活性位点辅因子是一个[(R - 高柠檬酸)MoFe₇S₉C]簇，被指定为M - 簇。这个重要的辅因子是由一对[FeS]簇逐步组装而成的，它们在进行进一步细化以完成生物合成之前先融合成一个[Fe₄S₄C]核心。NifB是自由基S - 腺苷 - L - 甲硫氨酸（SAM）超家族的成员，它促进了[FeS]簇（称为K - 簇）向[Fe₄S₄C]核心（称为L - 簇）的转化。这种转化包括一个依赖SAM的碳化物插入过程，同时还会掺入一个额外的硫原子。虽然过去NifB的纯化困难阻碍了详细的生化分析，但我们已经在大肠杆菌中开发了一种异源表达系统，该系统能从各种固氮产甲烷生物中产生稳定的NifB蛋白，可用于相关研究。本章详细介绍了制备活性NifB蛋白所需的步骤。还描述了用于SAM依赖的碳化物插入反应生化特性表征的方法。
