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Identification of a second site compensatory mutation in the Fe‐protein that allows diazotrophic growth of Azotobacter vinelandii UW97
Author(s) -
Lei Shi,
Pulakat Lakshmidevi,
Suh Man-Hee,
Gavini Narasaiah
Publication year - 2000
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/s0014-5793(00)01847-0
Subject(s) - azotobacter vinelandii , mutant , biochemistry , complementation , chemistry , serine , nitrogenase , diazotroph , mutation , nitrogen fixation , enzyme , organic chemistry , nitrogen , gene
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.