Research

Transcriptional regulator of nitrogenase, another pathway to understand nitorgenase

　In present days, artificial nitrogen fixation (reduction of nitrogen in the air to ammonia) reaches approximately to 10% of whole ecosystem (Ref. 1), which is essential for the production activities of the human race. Most of them have been demanded by the Haber process. In light of energy saving for material production, development of alternative processes that do not require high temperature and high pressure would become a more and more important issue (Ref. 1). Among several challenges to realize such processes, use of bacteria that produce nitrogenase (a nitrogen-fixing enzyme) as biomass is counted to be a strong candidate. However, there are several big problems before putting it into practical use. One of them is to understand what growing environment maximizes ability of bacterial nitrogen fixation. Results from a number of previous studies has indicated that the first phase of the functional regulation is done at a transcriptional level of the nitrogenase genes. Therefore, to understand the function regulation mechanism of nitrogenase, it would be essential to elucidate transcriptional regulation system of nitrogenase. In this study, we are aiming to elucidate the molecular mechanism of a transcriptional activator for nitrogenase from Azotobacter vinelandii (A. vinelandii) which is a well-studied diazotroph for many years.

A. vinelandii can grow in soil and water without being parasitic on roots of plants. Its genome has been well-analyzed and genetically engineered for many years. Thus, this bacterium has been considered to be one of the promising nitrogen-fixing bacteria for future practical use in biomass (Ref. 2). Genome analysis reported in 1989 for this bacterium revealed that there are three nitrogenase (1,2,3), each of which are under the regulation of specific transcriptional regulator as depicted in Figure 1. Since then, researches on a protein pair, NifA and NifL which serve to transcriptional control of nitrogenase-1 has been extensively carried out. According to Web of Science in October 2011, more than 100 reports have been published, which have contributed to reveal an environmental factor and its sensing mechanism in detail. On the other hand, with regard to VnfA, and AnfA, which are transcriptional activators of nitrogenase-2 and -3, less than 10 reports have been published until the same period. As the result, functional mechanisms of VnfA and AnfA were remained largely unclear at the time that we began this research. The biggest reason of such gap is that when expressed as a recombinant protein, they become insoluble and that it is extremely difficult to purify them. Actually, I was faced with the same problem when we started this study. Then, we focused on the Cys-rich motifs located on the N-terminal domains of the proteins (AnfA: -Cys8-X-Cys10-XXXX-Cys15-, VnfA: -Ser19-X-Cys21-XXXX-Cys26-), and anticipated that there must bind some metal ions or metal clusters there, otherwise the proteins get unstable resulting formation of inclusion bodies. Based on this idea, we introduced a Fe-S cluster biosynthesis machinery genes (suf genes) to E. coli to solubilize VnfA for subsequent purification. As a result, we succeeded for the first time to be obtained as soluble proteins VnfA which incorporates a 3Fe-4S cluster as a prosthetic group (Figure 2).

Figure 3. The elucidated functional mechanism of VnfA. See FEBS J. 2010, 2011 for details.

Figure 3. The elucidated functional mechanism of VnfA. See FEBS J. 2010, 2011 for details.
VnfA is the first example that harbors a 3Fe-4S cluster among all transcriptional regulators ever. Therefore, it must be interesting to understand its physiological significance.  Due to recent progresses, we are getting able to illustrate functional mechanism of VnfA as depiced in Figure 3.

 We believe that our study contributes to full understanding of the nitrogenase regulatory system in A. vinelandii and to put nitrogenases to a real system of "Fixing nitrogen by biomass" in near future.

References)

1. Nitrogen Fixation, 3rd Edition” John Postgate Ed., Cambridge University Press (1998).
2. Genetics and Regulation of Nitrogen Fixation in Free-Living Bacteria” Werner Klipp and Bernd Masepohl Ed., Kluwer Academic Publisher (2004).

Our paper concerned with this study）

1. The role of the GAF and central domains of the transcriptional activator VnfA in Azotobacter vinelandii, FEBS J. 2011, 278, 3287-3297, Yoshimitsu, K., Takatani, N., Kanematsu, Y., Miura, Y., Watanabe, Y., Nakajima, H.
   
   Nitrogenase transcription activator, VnfA is composed of three domains. Features of each domain had been expected from only homology analysis of the primary structure with well-elucidated proteins. Based on our purified VnfA, we experimentally elucidated functions of each domain qualitatively and quantitatively. The results allowed us to illustrate the overall mechanism to exert its transcriptional activity.
2. The role Fe-S cluster in the sensory domain of nitrogenase transcriptional activator VnfA Azotobacter vinelandii., FEBS J. 2010, 277, 817-832, Nakajima, H., Takatani, N., Yoshimitsu, K., Itoh, M., Aono, S., Takahashi, Y., Watanabe, Y.
   

Since the gene of VnfA was discovered in 1989 over 30 years, we successfully achieved for the first time to purified VnfA as a recombinant protein. By analyzing the purified protein, we obtained real answers about functional properties of VnfA expected at the genetic level. One of the highlights in this report is that this protein has a 3Fe-4S type cluster as a sensor moiety. Sensor proteins harboring 2Fe-2S and 4Fe-4S type clusters had been identified. This report finally added the 3Fe-4S type cluster to the sensing module of the sensor proteins, which was missing link in the function of Fe-S clusters.