Molecular and biochemical characterization of phosphoenolpyruvate carboxykinase in the ruminal bacterium Ruminococcus albus.
Molecular properties and transcriptional control of phosphoenolpyruvate carboxykinase (PCK; EC 4.1.1.32) in Ruminococcus albus were examined. The putative 537-amino acid PCK polypeptide has a predicted mass of 59.4 kDa and an isoelectric point of 4.82. RT-PCR and Northern blot analyses of pck mRNA suggest that the transcript is monocistronic and that pck transcription is not affected by changes in sugar sources present in growth medium. PCK enzymatic activity requires either Mg(2+) or Mn(2+) and an optimal pH of 7.0. R. albus PCK phosphorylated ADP more readily than GDP. Apparent K ( m ) values of PCK for PEP and ADP were considerably lower than those for OAA and ATP, suggesting that the reaction from PEP to OAA is favored in R. albus. The enzyme properties of PCK in R. albus appear to be more similar to Selenomonas ruminantium PCK than to Ruminococcus flavefacience, although R. albus and R. flavefacience belong to the same genus. The specific activity of PCK, representing the amount of enzyme per cell, in R. albus was much lower than that in S. ruminantium. The amount of succinate produced in R. albus from one unit of cellobiose was also much lower than the sum of succinate and propionate produced in S. ruminantium. Based on these results, we propose enhancement of PCK activity by stimulating PCK transcription as a method to decrease R. albus H(2) production without suppressing growth.
Asanuma
Narito
N
Department of Life Science, College of Agriculture, Meiji University, Higashimita, Kawasaki, Japan. asanuma@isc.meiji.ac.jp
Yoshizawa
Kimio
K
Kanada
Kazuhiro
K
Hino
Tsuneo
T
D000244:Adenosine Diphosphate / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D015152:Blotting, Northern; D002475:Cellobiose / Q000378:metabolism; D003067:Coenzymes / Q000494:pharmacology; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D020869:Gene Expression Profiling; D006153:Guanosine Diphosphate / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007526:Isoelectric Point; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D008345:Manganese / Q000494:pharmacology; D008969:Molecular Sequence Data; D008970:Molecular Weight; D019764:Phosphoenolpyruvate Carboxykinase (ATP) / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D045857:Ruminococcus / Q000201:enzymology* / Q000235:genetics*; D017422:Sequence Analysis, DNA; D013379:Substrate Specificity; D019802:Succinic Acid / Q000378:metabolism
D001426:Bacterial Proteins; D003067:Coenzymes; D004269:DNA, Bacterial; D006153:Guanosine Diphosphate; D002475:Cellobiose; D008345:Manganese; D000244:Adenosine Diphosphate; D019802:Succinic Acid; D019764:Phosphoenolpyruvate Carboxykinase (ATP); D008274:Magnesium