Posttranslational modification of Klebsiella pneumoniae flavodoxin by covalent attachment of coenzyme A, shown by 31P NMR and electrospray mass spectrometry, prevents electron transfer from the nifJ protein to nitrogenase. A possible new regulatory mechanism for biological nitrogen fixation.
A strain of Escherichia coli (71-18) that produces ca. 15% of its soluble cytoplasmic protein as a flavodoxin, the Klebsiella pneumoniae nifF gene product, has been constructed. The flavodoxin was purified using FPLC and resolved into two forms, designated KpFldI and KpFldII, which were shown to have identical N-terminal amino acid sequences (30 residues) in agreement with that predicted by the K. pneumoniae nifF DNA sequence. 31P NMR, electrospray mass spectrometry, UV-visible spectra, and thiol group estimations showed that the single cysteine residue (position 68) of KpFldI is posttranslationally modified in KpFldII by the covalent, mixed disulfide, attachment of coenzyme A. KpFldII was inactive as an electron carrier between the K. pneumoniae nifJ product (a pyruvate-flavodoxin oxidoreductase) and K. pneumoniae nifH product (the Fe-protein of nitrogenase). This novel posttranslational modification of a flavodoxin is discussed in terms of the regulation of nitrogenase activity in vivo in response to the level of dissolved O2 and the carbon status of diazotrophic cultures.
Thorneley
R N
RN
AFRC Institute of Plant Science Research, University of Sussex, Brighton, U.K.
Abell
C
C
Ashby
G A
GA
Drummond
M H
MH
Eady
R R
RR
Huff
S
S
Macdonald
C J
CJ
Shneier
A
A
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics*; D001483:Base Sequence; D003065:Coenzyme A / Q000378:metabolism*; D004261:DNA Replication; D004579:Electron Transport; D005418:Flavodoxin / Q000096:biosynthesis / Q000235:genetics*; D015964:Gene Expression Regulation, Bacterial; D005798:Genes, Bacterial; D007658:Ketone Oxidoreductases; D007711:Klebsiella pneumoniae / Q000201:enzymology / Q000235:genetics*; D009682:Magnetic Resonance Spectroscopy; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D009586:Nitrogen Fixation / Q000235:genetics*; D009591:Nitrogenase / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D013438:Sulfhydryl Compounds / Q000737:chemistry
D001426:Bacterial Proteins; D005418:Flavodoxin; D013438:Sulfhydryl Compounds; D009591:Nitrogenase; D007658:Ketone Oxidoreductases; C039443:pyruvate-flavodoxin oxidoreductase; D003065:Coenzyme A