Identification of a second site compensatory mutation in the Fe‐protein that allows diazotrophic growth of Azotobacter vinelandii UW97

FEBS Letters

Pulakat, Lakshmidevi¹  Lei, Shi¹  Gavini, Narasaiah¹  Suh, Man-Hee¹ 

[1] Department of Biological Sciences, Bowling Green State University, Bowling Green, OH 43403, USA

关键词: nifH;    Nitrogenase;    Fe-protein;    Second site mutation;    Azotobacter;   

DOI  :  10.1016/S0014-5793(00)01847-0

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

Azotobacter vinelandii UW97 is defective in nitrogen fixation due to a replacement of serine at position 44 by phenylalanine in the Fe-protein [Pulakat, L., Hausman, B.S., Lei, S. and Gavini, N. (1996) J. Biol. Chem. 271, 1884–1889]. Serine residue 44 is located in a conserved domain that links the nucleotide binding site and the MoFe-protein docking surface of the Fe-protein. Therefore, it is possible that the loss of function by A. vinelandii UW97-Fe-protein may be caused by global conformational disruption or disruption of the conformational change upon MgATP binding. To determine whether it is possible to generate a functional nitrogenase complex via a compensating second site mutation(s) in the Fe-protein, we have attempted to isolate genetic revertants of A. vinelandii UW97 that can grow on nitrogen-free medium. One such revertant, designated A vinelandii BG9, encoded a Fe-protein that retained the Ser44Phe mutation and also had a second mutation that caused the replacement of a lysine at position 170 by glutamic acid. Lysine 170 is highly conserved and is located in a conserved region of the Fe-protein. This region is implicated in stabilizing the MgATP-induced conformation of the Fe-protein and in docking to the MoFe-protein. Further complementation analysis showed that the Fe-protein mutant that retained serine 44 but contained the substitution of lysine at position 170 by glutamic acid was also non-functional. Thus, neither Ser44Phe nor Lys170Glu mutants of Fe-protein were functional; however, the Fe-protein in A. vinelandii BG9 that contained both substitutions could support diazotrophic growth on the strain.

【 授权许可】

Unknown