Synthesis of ATP derivatives of compounds of the mevalonate pathway (isopentenyl di- and triphosphate; geranyl di- and triphosphate, farnesyl di- and triphosphate, and dimethylallyl diphosphate) catalyzed by T4 RNA ligase, T4 DNA ligase and other ligases

Abstract

Compounds of the mevalonate pathway containing a terminal di- or triphosphate (mev-PP or mev-PPP) were tested as substrates of several enzyme ligases (T4 RNA ligase, T4 DNA ligase, firefly luciferase and other ligases) for the synthesis of ATP derivatives of the mev-pppA or mev-ppppA type. T4 RNA ligase, in the presence of ATP and the substrates: geranyl, farnesyl or isopentenyl triphosphates, and geranyl, farnesyl, dimethylallyl or isopentenyl diphosphates, all at 0.3 mM concentration, catalyzed the synthesis of the corresponding ATP derivatives at a relative rate of activity of: 7.6+/-1.4 mU/mg or 100%; 39%; 42%; 24%; 18%; 12% and 6%, respectively. Inhibition (%) of the synthesis by excess of substrate (0.8 mM vs. 0.3 mM) was observed with farnesyl diphosphate (99%); farnesyl triphosphate (96%) and geranyl triphosphate (32%). V(max), K(m), K(cat) and K(cat)/K(m) values were also determined. The K(cat)/K(m) values calculated were for: farnesyl triphosphate, 166; geranyl triphosphate, 52.2; farnesyl diphosphate, 12.1; geranyl diphosphate, 8.6; isopentenyl triphosphate, 6.7; dimethylallyl diphosphate, 3.1 and isopentenyl diphosphate, 0.9. Similar results were obtained with T4 DNA ligase. The above-mentioned compounds were also substrates of firefly luciferase synthesizing the mev-pppA or mev-ppppA derivatives. In our hands, neither the acyl- or acetyl-CoA synthetases nor the ubiquiting activating enzyme (E1) catalyzed the synthesis of ATP derivatives of these compounds. The results here presented could be related with the mechanism of action of bisphosphonates on osteoclasts or tumor cells.

Purinergic signalling in the musculoskeletal system

“…The antitumour effects of these drugs are not clear but could involve apoptosis induced by the ATP analogue ApppI produced by nitrogen-containing bisphosphonates [453,454].…”

Effect of Bisphosphonates on Root Growth and on Chlorophyll Formation in Arabidopsis thaliana Seedlings

“…Following this finding, the synthesis of derivatives of a variety of compounds of the mevalonate pathway capped with an adenosine moiety catalyzed by several ligases, was later reported (mev-pppA and mev-ppppA) (Günther Sillero et al, 2009). Increase in the concentration of metabolites upstream the inhibited step could stimulate synthesis of the corresponding mevalonate derivative (reaction 3) (Günther Sillero et al, 2009;Rogers et al, 2010).…”

“…As in these reactions BPs act as analogs of PP it would be expected that the non-N-BP (the smallest type of BP) were the preferred substrates for these reactions. b) BPs may be inhibitors of enzymes having substrates with a terminal PP (R-PP) (see below and Günther Sillero et al, 2009). Although the inhibition of these enzymes could take place at any step of the pathway, specific inhibitions have been reported on isolated enzymes from bacteria, yeast or plants: pyrophosphatase (Baykov et al, 1993;Cromartie et al, 1999;Drozdowicz et al, 2003;Gordon-Weeks et al, 1999;Kim et al,1994;Kuo et al, 2005;Rodrígues et al, 2000;Szabo & Oldfield, 2001;Zhen et al, 1994 ); geranyl diphospho synthetase (Burke et al, 2004;Oberhauser et al, 1998); isopentenyl pyrophosphate synthase (Cromartie et al, 1999) and P5C reductase (Forlani et al, 2008).…”

“…Following this finding, the synthesis of derivatives of a variety of compounds of the mevalonate pathway capped with an adenosine moiety catalyzed by several ligases, was later reported (mev-pppA and mev-ppppA) (Günther Sillero et al, 2009). Increase in the concentration of metabolites upstream the inhibited step could stimulate synthesis of the corresponding mevalonate derivative (reaction 3) (Günther Sillero et al, 2009;Rogers et al, 2010). Related to the use of bisphosphonates in humans for the treatment of osteoporosis, Paget's disease and bone tumour metastasis it can be stressed that upon their oral or intravenous administration BP are partially eliminated by kidney and partially fixed in bones, with very little amount in the systemic circulation (Cremers et al, 2005).…”

“…This later character, used as an indicator of the ability of plants for performing the fundamental process of photosynthesis, also indicates the alteration of the mevalonate pathway, since chlorophyll is a product of this biosynthetic pathway. We have focused our attention on this pathway as a good target to examine the effect of BP given the two metabolic peculiarities present only in the mevalonate cycle: occurrence of both three consecutive enzymes requiring ATP as a cosubstrate and the major pool of compounds containing a terminal pyrophosphate in any known metabolic pathway (Günther Sillero et al, 2009) (Fig. 1).…”