The major iron-containing protein of Legionella pneumophila is an aconitase homologous with the human iron-responsive element-binding protein.
Legionella pneumophila has high iron requirements, and its intracellular growth in human monocytes is dependent on the availability of intracellular iron. To learn more about iron metabolism in L. pneumophila, we have undertaken an analysis of the iron proteins of the bacterium. We first developed an assay to identify proteins by 59Fe labelling and nondenaturing polyacrylamide gel electrophoresis. The assay revealed seven iron proteins (IPs) with apparent molecular weights of 500, 450, 250, 210, 150, 130, and 85. IP150 comigrates with superoxide dismutase activity and is probably the Fe-superoxide dismutase of L. pneumophila. IP210 is the major iron-containing protein (MICP). To identify and characterize MICP, we purified the protein and cloned and sequenced its gene. MICP is a monomeric protein containing 891 amino acids, and it has a calculated molecular mass of 98,147 Da. Analysis of the sequence revealed that MICP has two interesting homologies. First, MICP is highly homologous with the human iron-responsive element-binding protein, consistent with the hypothesis that this critical iron-regulatory molecule of humans has a prokaryotic ancestor. Second, MICP is highly homologous with the Escherichia coli aconitase and to a lesser extent with porcine heart mitochondrial aconitase. Consistent with this, we found that MICP exhibits aconitase activity. In contrast to other aconitases, MICP has a single amino acid change of a potentially deleterious type at a site thought to be critical for substrate binding and enzymatic activity. However, the specific activity of MICP is roughly comparable to that of other aconitases, suggesting that the mutation has at most a mild effect on the aconitase activity of MICP. The abundance of MICP in L. pneumophila suggests either that L. pneumophila requires high aconitase and perhaps tricarboxylic acid cycle activity or that the bacterium requires large amounts of this protein to serve an additional role in bacterial physiology. A need for large amounts of MICP, which contains four Fe atoms per molecule when fully loaded, could at least partly explain L. pneumophila's high metabolic requirement for iron.
Mengaud
J M
JM
Department of Medicine, School of Medicine, University of California, Los Angeles 90024.
Horwitz
M A
MA
D000154:Aconitate Hydratase / Q000737:chemistry* / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D001483:Base Sequence; D003001:Cloning, Molecular; D004269:DNA, Bacterial; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D007501:Iron / Q000737:chemistry* / Q000378:metabolism; D035925:Iron-Regulatory Proteins; D016952:Legionella pneumophila / Q000201:enzymology*; D008969:Molecular Sequence Data; D016601:RNA-Binding Proteins / Q000737:chemistry* / Q000302:isolation & purification; D015183:Restriction Mapping; D017386:Sequence Homology, Amino Acid; D013552:Swine
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D035925:Iron-Regulatory Proteins; D016601:RNA-Binding Proteins; D007501:Iron; C082937:ACN protein, Legionella pneumophila; D000154:Aconitate Hydratase