4-Coumarate:Coenzyme A Ligase Has the Catalytic Capacity to Synthesize and Reuse Various (Di)Adenosine Polyphosphates

Abstract

4-Coumarate:coenzyme A ligase (4CL) is known to activate cinnamic acid derivatives to their corresponding coenzyme A esters. As a new type of 4CL-catalyzed reaction, we observed the synthesis of various mono-and diadenosine polyphosphates. Both the native 4CL2 isoform from Arabidopsis (At4CL2 wild type) and the At4CL2 gain of function mutant M293P/K320L, which exhibits the capacity to use a broader range of phenolic substrates, catalyzed the synthesis of adenosine 5Ј-tetraphosphate (p 4 A) and adenosine 5Ј-pentaphosphate when incubated with MgATP Ϫ2 and tripolyphosphate or tetrapolyphosphate (P 4 ), respectively. Diadenosine 5Ј,5ٞ,-P 1 ,P 4 -tetraphosphate represented the main product when the enzymes were supplied with only MgATP 2Ϫ . The At4CL2 mutant M293P/K320L was studied in more detail and was also found to catalyze the synthesis of additional dinucleoside polyphosphates such as diadenosine 5Ј,5ٞ-P 1 ,P 5 -pentaphosphate and dAp 4 dA from the appropriate substrates, p 4 A and dATP, respectively. Formation of Ap 3 A from ATP and ADP was not observed with either At4CL2 variant. In all cases analyzed, (di)adenosine polyphosphate synthesis was either strictly dependent on or strongly stimulated by the presence of a cognate cinnamic acid derivative. The At4CL2 mutant enzyme K540L carrying a point mutation in the catalytic center that is critical for adenylate intermediate formation was inactive in both p 4 A and diadenosine 5Ј,5ٞ,-P 1 ,P 4 -tetraphosphate synthesis. These results indicate that the cinnamoyl-adenylate intermediate synthesized by At4CL2 not only functions as an intermediate in coenzyme A ester formation but can also act as a cocatalytic AMP-donor in (di)adenosine polyphosphate synthesis.

Response of barley seedlings to water deficit and enhanced UV-B irradiation acting alone and in combination

“…At the lack of coenzyme A, 4:coumarate-CoA ligase may catalyze the synthesis of nucleotides such as adenosine-5 0 -tetraphosphate and diadenosine tetraphosphate, whose functions in plants have not been clarified to date (Pietrowska-Borek et al 2003). It could have occurred in the roots of barley plants treated with UV-B radiation in this study, where a considerable increase was observed in the activity of 4CL and a lack of that of anthocyanins and flavonols.…”

The substrate specificity-determining amino acid code of 4-coumarate:CoA ligase

“…In addition, several of these secondary plant products have been suggested to have a health-promoting function in human nutrition (3). Recently, the in vitro synthesis of various monoadenosine and diadenosine polyphosphates in the absence of CoA has been observed as an additional 4CL-catalyzed reaction (4). However, the biological significance of this second 4CL-associated reaction is still unknown, although the accumulation of (di)adenosine polyphosphates has been correlated with stress responses in bacteria and fungi and cell differentiation and apoptosis in cultured mammalian cells (5,6).…”

Exogenous adenosine 5′-phosphoramidate behaves as a signal molecule in plants; it augments metabolism of phenylpropanoids and salicylic acid in Arabidopsis thaliana seedlings

“…In particular, dinucleoside polyphosphates, including diadenosine tri-and tetraphosphates (ApppA and AppppA), can be synthesized by certain ligases (Zamecnik et al, 1966;Goerlich et al, 1982;Jakubowski, 1983;Pietrowska-Borek et al, 2003) and transferases (Wang and Shatkin, 1984;Guranowski et al, 1988Guranowski et al, , 2004. There are also different specific and nonspecific enzymes that catalyze the hydrolytic or phosphorolytic degradation of these compounds (for review see: Guranowski, 2000).…”