Identification of a second site compensatory mutation in the Fe-protein that allows diazotrophic growth of Azotobacter vinelandii UW97.
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.
Department of Biological Sciences, Bowling Green State University, 43403, Bowling Green, OH, USA.
S
S
Lei
L
L
Pulakat
M
M
Suh
N
N
Gavini
D000255:Adenosine Triphosphate / Q000378:metabolism; D019943:Amino Acid Substitution / Q000235:genetics; D016948:Azotobacter vinelandii / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development* / Q000378:metabolism; D017124:Conserved Sequence / Q000235:genetics; D008958:Models, Molecular; D008983:Molybdoferredoxin / Q000378:metabolism; D009584:Nitrogen / Q000378:metabolism; D010088:Oxidoreductases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D010641:Phenotype; D011487:Protein Conformation; D013489:Suppression, Genetic / Q000235:genetics*
D008983:Molybdoferredoxin; D000255:Adenosine Triphosphate; D010088:Oxidoreductases; C029130:nitrogenase reductase; D009584:Nitrogen