Francisco Javier Pérez-Zúñiga

Synthesis of bisphosphonate derivatives of ATP by T4 RNA ligase

T4 RNA ligase catalyzes the synthesis of ATP β,γ‐bisphosphonate analogues, using the following substrates with the relative velocity rates indicated between brackets: methylenebisphosphonate (pCH2p) (100), clodronate (pCCl2p) (52), and etidronate (pC(OH)(CH3)p) (4). The presence of pyrophosphatase about doubled the rate of these syntheses. Pamidronate (pC(OH)(CH2–CH2–NH2)p), and alendronate (pC(OH)(CH2–CH2–CH2–NH2)p) were not substrates of the reaction. Clodronate displaced the AMP moiety of the complex E‐AMP in a concentration dependent manner. The K m values and the rate of synthesis (k cat) determined for the bisphosphonates as substrates of the reaction were, respectively: methylenebisphosphonate, 0.26 ± 0.05 mM (0.28 ± 0.05 s−1); clodronate, 0.54 ± 0.14 mM (0.29 ± 0.05 s−1); and etidronate, 4.3 ± 0.5 mM (0.028 ± 0.013 s−1). In the presence of GTP, and ATP or AppCCl2p the relative rate of synthesis of adenosine 5′,5‴‐P1,P4‐tetraphosphoguanosine (Ap4G) was around 100% and 33%, respectively; the methylenebisphosphonate derivative of ATP (AppCH2p) was a very poor substrate for the synthesis of Ap4G. To our knowledge this report describes, for the first time, the synthesis of ATP β,γ‐bisphosphonate analogues by an enzyme different to the classically considered aminoacyl‐tRNA synthetases.

Bisphosphonates activate the 5-fluorouracil/uracil phosphoribosyltransferase activity present in Saccharomyces cerevisiae cell extracts: Implications for tumor treatments

Most of the effects described for bisphosphonates (pC(R1)(R2)p) are related, directly or indirectly with a pyrophosphate moiety. Bisphosphonates are (i) analogs of pyrophosphate in the synthesis of ATP derivatives (AppC(R1)(R2)p) catalyzed by ligases and (ii) inhibitors of enzymes of the mevalonate pathway with substrates containing a terminal pyrophosphate. Searching for the role of bisphosphonates on other reactions involving pyrophosphate, we explored their effect on a phosphoribosyltransferase activity, present in Saccharomyces cerevisiae cell extracts, using 5-fluorouracil or uracil as substrates. Unexpectedly, bisphosphonates increased the initial rate of synthesis of 5-FUMP (from 5-fluorouracil and phosphoribosylpyrophosphate): etidronate (2.8+/-0.3 times); pamidronate (2.6+/-0.4 times); alendronate (2.5+/-0.6 times) and clodronate (2.0+/-0.1 times). Similar values for the synthesis of UMP (from uracil and phosphoribosylpyrophosphate) were obtained in the presence of bisphosphonates. The values of the activation constants determined for alendronate and clodronate for the synthesis of UMP were 0.05+/-0.02 mM and 0.32+/-0.22 mM, respectively. These results raise the possibility that bisphosphonates enhance the effect of 5-fluorouracil (or other uracil prodrugs) in the treatment of bone tumors or bone tumor metastases.

Metabolites produced during fermentation of wine by mixed cultures of yeasts and lactic acid bacteria

ZusammenfassungEs wurden Wechselbeziehungen zwischen Hefen und Milchsäurebakterien während der Fermentation von Traubensaft untersucht.Saccharomyces pastorianus bewirkte ein Ansteigen der Ketosäurenproduktion und der Bildung von Rest-Metaboliten. Das Hinzufügen vonLactobacillus plantarum beim Gärungsprozess kann die alkoholische Gärung unterdrücken und hat die Produktion von Acetaldehyd sowie vonl(+)- undd(−)-Milchsäuren beträchtlich gesteigert.AbstractCertain aspects of the interrelationship between associations of yeasts and lactic acid bacteria during the fermentation of grape must were studied. Action bySaccharomyces pastorianus gave rise to increased formation of keto acids and residual metabolites. Addition ofLactobacillus plantarum to the fermentation process may inhibit alcoholic fermentation and considerably increased the production of acetaldehyde andl(+) andd(−)lactic acids.