A hybrid Azotobacter vinelandii-Clostridium pasteurianum nitrogenase iron protein that has in vivo and in vitro catalytic activity.
Site-directed mutagenesis and gene replacement procedures were used to construct a mutant strain of Azotobacter vinelandii which expresses a hybrid nitrogenase Fe protein. This hybrid Fe protein has its carboxyl-terminal 18 residues replaced with the 5 analogous residues from the Clostridium pasteurianum Fe protein sequence. The hybrid Fe protein is 13 amino acids smaller than the wild-type A. vinelandii Fe protein and has a net loss of 4 negatively charged residues, resulting in a change in size and charge. The strain which produces the hybrid Fe protein remained capable of diazotrophic growth, albeit at a reduced rate. Also, the purified hybrid Fe protein exhibited a maximum activity about one-half that of native Fe protein. These results demonstrate that the tight, inactive complex which is formed when A. vinelandii MoFe protein and C. pasteurianum Fe protein are mixed in heterologous reconstitution experiments cannot be accounted for only by differences in the A. vinelandii and C. pasteurianum Fe protein primary sequences located at their respective carboxyl termini.
Jacobson
M R
MR
Department of Anaerobic Microbiology, Virginia Polytechnic Institute and State University, Blacksburg 24061.
Cantwell
J S
JS
Dean
D R
DR
D001395:Azotobacter / Q000201:enzymology*; D001483:Base Sequence; D002384:Catalysis; D003001:Cloning, Molecular; D003013:Clostridium / Q000201:enzymology*; D007501:Iron; D008969:Molecular Sequence Data; D008982:Molybdenum; D016297:Mutagenesis, Site-Directed; D009591:Nitrogenase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D055503:Protein Multimerization; D011994:Recombinant Proteins / Q000378:metabolism; D015183:Restriction Mapping; D014169:Transformation, Bacterial
D011994:Recombinant Proteins; D008982:Molybdenum; D007501:Iron; D009591:Nitrogenase