Molecular characterization, enzyme properties and transcriptional regulation of phosphoenolpyruvate carboxykinase and pyruvate kinase in a ruminal bacterium, Selenomonas ruminantium

elucidate the regulatory mechanism for propionate production in Selenomonas ruminantium, the molecular properties and gene expression of phosphoenolpy ruvate carboxykinase (Pck) and pyruvate kinase (Pyk) were investigated, The Pck was deduced to consist of 538 aa with a molecular mass of 59.6 kDa, an d appeared to exist as a monomer. The Pyk was revealed to consist of four i dentical subunits consisting of 469 aa with a molecular mass of 51.3 kDa. B oth Mg2+ and Mn2+ were required for the maximal activity of Pck, and Pck ut ilized ADP, not CDP or IDP, as a substrate. Either Mg2+ or Mn2+ was require d for Pyk activity, and the enzyme was activated by phosphoenolpyruvate (PE P) and fructose 1,6-bisphosphate (FBP). Pyk activity was severely inhibited by P-i, but restored by the addition of FBP. The K-m value of Pck for PEP (0.55 mM) was nearly equal to the K-m value of Pyk for PEP, suggesting that the partition of the flow from PEP in the fermentation pathways is determi ned by the activity ratio of Pck to Pyk. Both pck and pyk genes were monoci stronic, although two transcriptional start sites were found in pyk. The le vel of pyk mRNA was not different whether glucose or lactate was the energy substrate. However, the pck mRNA level was 12-fold higher when grown on la ctate than on glucose. The level of pck mRNA was inversely related to the s ufficiency of energy, suggesting that Pck synthesis is regulated at the tra nscriptional level when energy supply is altered. It was conceivable that t he transcription of pck in S. ruminantium is triggered by PEP and suppresse d by ATP.