Molecular characterization and expression of pyruvate formate-lyase-activating enzyme in a ruminal bacterium, Streptococcus bovis.
To clarify the significance of the activation of pyruvate formate-lyase (PFL) by PFL-activating enzyme (PFL-AE) in Streptococcus bovis, the molecular properties and gene expression of PFL-AE were investigated. S. bovis PFL-AE was deduced to consist of 261 amino acids with a molecular mass of 29.9 kDa and appeared to be a monomer protein. Similar to Escherichia coli PFL-AE, S. bovis PFL-AE required Fe(2+) for activity. The gene encoding PFL-AE (act) was found to be polycistronic, and the PFL gene (pfl) was not included. However, the act mRNA level changed in parallel with the pfl mRNA level, responding to growth conditions, and the change was contrary to the change in the lactate dehydrogenase (LDH) mRNA level. PFL-AE synthesis appeared to change in parallel with PFL synthesis. Introduction of a recombinant plasmid containing S. bovis pfl and the pfl promoter into S. bovis did not affect formate and lactate production, which suggests that the activity of the pfl promoter is low. When the pfl promoter was replaced by the S. bovis ldh promoter, PFL was overexpressed, which caused an increase in the formate-to-lactate ratio. However, when PFL-AE was overexpressed, the formate-to-lactate ratio did not change, suggesting that PFL-AE was present at a level that was high enough to activate PFL. When both PFL-AE and PFL were overexpressed, the formate-to-lactate ratio further increased. It is conceivable that LDH activity is much higher than PFL activity, which may explain why the formate-to-lactate ratio is usually low.
Asanuma
Narito
N
Department of Life Science, College of Agriculture, Meiji University, Higashimita, Tama-ku, Kawasaki 214-8571, Japan.
Hino
Tsuneo
T
D000123:Acetyltransferases / Q000378:metabolism*; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D004789:Enzyme Activation; D004798:Enzymes / Q000235:genetics / Q000378:metabolism*; D005561:Formates / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial; D019344:Lactic Acid / Q000378:metabolism; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000378:metabolism; D016415:Sequence Alignment; D016985:Streptococcus bovis / Q000201:enzymology* / Q000235:genetics; D014158:Transcription, Genetic
D001426:Bacterial Proteins; D004798:Enzymes; D005561:Formates; D011994:Recombinant Proteins; C088618:lipase activator protein, Bacteria; C030544:formic acid; D019344:Lactic Acid; D000123:Acetyltransferases; C040285:pyruvate formate-lyase activating enzyme; C022560:formate C-acetyltransferase