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

Department of Life Science, College of Agriculture, Meiji University, Higashimita, Tama-ku, Kawasaki 214-8571, Japan¹

Author for correspondence: Tsuneo Hino. Tel: +81 44 934 7825. Fax: +81 44 934 7825. e-mail: hino{at}isc.meiji.ac.jp

To elucidate the regulatory mechanism for propionate production in Selenomonas ruminantium, the molecular properties and gene expression of phosphoenolpyruvate 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, and appeared to exist as a monomer. The Pyk was revealed to consist of four identical subunits consisting of 469 aa with a molecular mass of 51·3 kDa. Both Mg²⁺ and Mn²⁺ were required for the maximal activity of Pck, and Pck utilized ADP, not GDP or IDP, as a substrate. Either Mg²⁺ or Mn²⁺ was required for Pyk activity, and the enzyme was activated by phosphoenolpyruvate (PEP) 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 determined by the activity ratio of Pck to Pyk. Both pck and pyk genes were monocistronic, although two transcriptional start sites were found in pyk. The level 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 lactate than on glucose. The level of pck mRNA was inversely related to the sufficiency of energy, suggesting that Pck synthesis is regulated at the transcriptional level when energy supply is altered. It was conceivable that the transcription of pck in S. ruminantium is triggered by PEP and suppressed by ATP.

Keywords: Selenomonas ruminantium, phosphoenolpyruvate carboxykinase, pyruvate kinase, propionate

Abbreviations: Pck, phosphoenolpyruvate carboxykinase; Pyk, pyruvate kinase; PEP, phosphoenolpyruvate; OAA, oxaloacetate; FBP, fructose 1,6-bisphosphate; Ldh, lactate dehydrogenase

The GenBank accession numbers for the S. ruminantium pck and pyk sequences reported in this paper are AB016600 and AB037182, respectively.

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