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Analysis of site‐directed mutations in the α‐and β‐subunits of Klebsiella pneumoniae nitrogenase
Author(s) -
Kent H. M.,
Baines M.,
Gormal C.,
Smith B. E.,
Buck M.
Publication year - 1990
Publication title -
molecular microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.857
H-Index - 247
eISSN - 1365-2958
pISSN - 0950-382X
DOI - 10.1111/j.1365-2958.1990.tb02060.x
Subject(s) - biology , nitrogenase , mutant , alanine , cysteine , biochemistry , protein subunit , mutagenesis , site directed mutagenesis , amino acid , tyrosine , polyclonal antibodies , gel electrophoresis , klebsiella pneumoniae , wild type , microbiology and biotechnology , escherichia coli , enzyme , bacteria , gene , nitrogen fixation , genetics , antibody
Summary Using directed mutagenesis, amino acid substitutions have been made in the α‐ and β‐subunits of the Klebsiella pneumoniae nitrogenase component 1 at positions normally occupied by conserves cysteine or tyrosine residues. Nif', Nif and intermediate pheno‐types have been obtained. To extend our earlier biochemical characterization (Kent et al , 1989) the electrophoretic mobiliy of component 1 of the mutant and wild‐type nitrogenases has been analysed by non‐denaturing gel electrophoresis. The major and minor forms of component 1 separated by this methodology have been probed for by using both polyclonal and monoclonal antibodies. All Nif mutants exhibited a distribution of electrophoretic forms of component 1 comparable to the wild type, and the abundance of the major from found in purified nitrogennase correlated approximately with the specific activity of the extract. In contrast, after electrophoresis, component 1 from Nif mutants exhibited either a major low‐mobility from or a fast‐moving from. Analysis of co‐factor (FeMoco) allowed us to conclude that changing cysteine 275 to alanine in the α‐submit produces component 1 defective in its interaction with FeMoco. Substitution of other con‐served cysteine residues by alanine appears to prevent early steps in nitrogebnase assembly or to promote degradation. Two single mutations (cysteine89 to alanine in the β‐subunit) which are tightly Nif can be combined to produce a weakly active nitrogenase, indicating regions involved in the interaction between subunits.