Effects of pH and Energy Supply on Activity and Amount of Pyruvate Formate-Lyase in Streptococcus bovis

Abstract

The enzyme system of pyruvate formate-lyase (PFL) in Streptococcus bovis was investigated by isolating PFL and PFL-activating enzyme (PFL-AE) from S. bovis, flavodoxin from Escherichia coli, and chloroplasts from spinach. In this study, the PFL and PFL-AE in S. bovis were found to be similar to those in E. coli, suggesting that the activating mechanisms are similar. The optimal pH of S. bovis PFL was 7.5, which is in contrast to the optimal pH of S. bovis lactate dehydrogenase, which is 5.5. The apparent K m of S. bovis PFL was 2 mM. The intermediates of glycolysis, dihydroxyacetone phosphate (DHAP) and D-glyceraldehyde-3-phosphate (GAP), were shown to inhibit PFL activity. The concentrations of intracellular DHAP and GAP in S. bovis ranged from 1.9 mM to less than 0.1 mM and from 0.6 mM to less than 0.05 mM, respectively, depending on the energy supply. The wide variations in DHAP and GAP levels indicated that PFL activity is allosterically regulated by these triose phosphates in vivo. The amount of PFL protein, as determined by Western blot analysis with polyclonal antibody, changed in parallel with the level of pfl-mRNA, responding to the culture conditions. These observations confirm that PFL synthesis is regulated at the transcriptional level and support the hypothesis that S. bovis shifts the fermentation pathway from acetate, formate, and ethanol production to lactate production when the pH is low and when excess energy is supplied.Streptococcus bovis is one of the prevailing microbes in the rumen, especially in animals receiving a high-starch diet. Its primary fermentation product is lactate (16,19). In this organism, pyruvate is either converted to lactate by lactate dehydrogenase (LDH) or converted to acetyl coenzyme A and formate by pyruvate formate-lyase (PFL) (19) and the acetyl coenzyme A is then converted either to acetate or ethanol. The percentages of lactate in fermentation products differ under different growth conditions, and the percentage is determined by the relative activities of LDH and PFL (2,4,19).We previously reported that S. bovis increases the percentage of lactate by increasing the amount of LDH in response to low pH and an excess energy supply (2-4) and that LDH synthesis is regulated at the transcriptional level (3, 4). The increase in lactate production at low pH is explained in part by the fact that the optimal pH of S. bovis LDH is 5.5, which is similar to the intracellular pH when the extracellular pH is 4.7 (2, 19). The enhanced lactate production in the presence of an excess energy supply can be explained in part by the activation of LDH by an increased concentration of intracellular fructose-1,6-bisphosphate (2, 19). Thus, lactate production by S. bovis is regulated by the ultimate activity of LDH, which includes the amount of enzyme protein and the function of the enzyme.When the pH was low and the energy supply was in excess, the level of pfl-mRNA decreased, while the level of ldh-mRNA increased (4). The activity of PFL decreased with a decrease in the level of pfl-mRNA...

Effects of the overexpression of fructose-1,6-bisphosphate aldolase on fermentation pattern and transcription of the genes encoding lactate dehydrogenase and pyruvate formate-lyase in a ruminal bacterium, Streptococcus bovis

“…After being centrifuged again, the supernatant was immediately subjected to analysis. The activities of FBA, LDH, and PFL were assayed by the method described previously Hino, 1997, 2002a;Asanuma et al, 1999Asanuma et al, , 2000. Activities of FBA and PFL were measured by coupled enzyme assays with glycerol-3-phosphate dehydrogenase and malate dehydrogenase plus citrate synthase, respectively.…”

“…For example, LDH is activated by fructose-1,6-bisphosphate (FBP), and PFL is inhibited by dihydroxyacetone phosphate (DHAP) and D-glyceraldehyde-3-phosphate (GAP). The other is transcriptional control of enzyme synthesis (Asanuma and Hino, 2000;Asanuma et al, , 1999. For example, when S. bovis was grown at low pH or under excess-energy conditions, the LDHmRNA level increased and the PFL-mRNA level decreased (Asanuma and Hino, 2000;Asanuma et al, , 1999.…”

“…The other is transcriptional control of enzyme synthesis (Asanuma and Hino, 2000;Asanuma et al, , 1999. For example, when S. bovis was grown at low pH or under excess-energy conditions, the LDHmRNA level increased and the PFL-mRNA level decreased (Asanuma and Hino, 2000;Asanuma et al, , 1999. This suggests that S. bovis alters the amounts of LDH and PFL in response to growth conditions.…”

“…The change is primarily brought about at the first branch point of fermentation pathway, pyruvate. Increase in lactate production can be explained by an increase in lactate dehydrogenase (LDH) activity and a simultaneous decrease in pyruvate-formate lyase (PFL) activity (Asanuma and Hino, 2000;Asanuma et al, , 1999.…”

Properties and Regulation of the Bifunctional Enzyme HPr Kinase/Phosphatase in Bacillus subtilis

“…FBP was initially tested at 2 mM, a concentration found in B. subtilis when grown on D-glucose (58), and F2,6BP was also initially tested at a similar concentration, 1 mM. DL-Glyceraldehyde 3-phosphate was included at a concentration of 1 mM, since intracellular concentrations of this intermediate were found to be as high as 0.6 mM in Streptoccus bovis (59). Acetyl coenzyme A and acetyl phosphate were tested at concentrations of 0.5 and 1 mM, respectively.…”