Structure-function relationships in the alpha subunit of Klebsiella pneumoniae nitrogenase MoFe protein from analysis of nifD mutants.
Crude extracts of wild-type, nitrogenase-derepressed Klebsiella pneumoniae fractionated by nondenaturing gel electrophoresis contain, in addition to the major form of the MoFe protein, two minor variants of lower electrophoretic mobility. Of seven Nif- mutants of K. pneumoniae with nonpolar point mutations in nifD (encoding the alpha subunit of Kp1), three exhibit a wild-type-like electrophoretic pattern, whereas in the remaining four, the slowest-migrating form becomes the predominant species. Amino acid substitutions in mutants of the first type are located in the N terminus of NifD and include Gly-85 to Arg (UN1661), Glu-121 to Lys (UN1649), and Gly-161 to Asp (UN1683). Mutations of the second type are Gly-186 to Asp (UN1648), Gly-195 to Glu (UN1680), Ser-443 to Pro (UN1793), and Gly-455 to Asp (UN1650). Six of the mutated residues show interspecies conservation, three are close to conserved cysteines, and two are located next to conserved histidines. Based on evidence pointing to the possibility that the lowest-mobility form lacks the iron-molybdenum cofactor, these results provide insights into the functional significance of specific sites in the alpha subunit of the MoFe protein.
Govezensky
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Biochemistry Department, Weizmann Institute of Science, Rehovot, Israel.
Zamir
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A
D000595:Amino Acid Sequence; D000942:Antigens, Bacterial / Q000235:genetics; D003001:Cloning, Molecular; D004252:DNA Mutational Analysis; D015180:Electrophoresis, Gel, Two-Dimensional; D005798:Genes, Bacterial; D007711:Klebsiella pneumoniae / Q000201:enzymology* / Q000235:genetics; D008667:Metalloproteins / Q000502:physiology / Q000648:ultrastructure*; D008969:Molecular Sequence Data; D008982:Molybdenum; D009586:Nitrogen Fixation / Q000235:genetics; D009591:Nitrogenase / Q000235:genetics / Q000276:immunology; D013329:Structure-Activity Relationship
D000942:Antigens, Bacterial; D008667:Metalloproteins; D008982:Molybdenum; D009591:Nitrogenase