Plant Physiol, March 2003, Vol. 131, pp. 1401-1410

4-Coumarate:Coenzyme A Ligase Has the Catalytic Capacity to Synthesize and Reuse Various (Di)Adenosine Polyphosphates¹

Magorzata Pietrowska-Borek, Hans-Peter Stuible, Erich Kombrink, and Andrzej Guranowski^*

Katedra Biochemii i Biotechnologii, Akademia Rolnicza, ul. Woyska 35, 60-637 Pozna, Poland (M.P.-B., A.G.); and Max-Planck-Institut für Züchtungsforschung, Abteilung Biochemie, Carl-von-Linné-Weg 10, 50829 Köln, Germany (H.-P.S., E.K.)

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 5'-tetraphosphate (p₄A) and adenosine 5'-pentaphosphate when incubated with MgATP² and tripolyphosphate or tetrapolyphosphate (P₄), respectively. Diadenosine 5',5,-P¹,P⁴-tetraphosphate represented the main product when the enzymes were supplied with only MgATP². 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 5',5-P¹,P⁵-pentaphosphate and dAp₄dA from the appropriate substrates, p₄A and dATP, respectively. Formation of Ap₃A 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 p₄A and diadenosine 5',5,-P¹,P⁴-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.