4-Coumarate:coenzyme A ligase has the catalytic capacity to synthesize and reuse various (di)adenosine polyphosphates.
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 (p(4)A) and adenosine 5'-pentaphosphate when incubated with MgATP(-2) and tripolyphosphate or tetrapolyphosphate (P(4)), respectively. Diadenosine 5',5''',-P(1),P(4)-tetraphosphate represented the main product when the enzymes were supplied with only MgATP(2-). 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'''-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 5',5''',-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.
Pietrowska-Borek
Małgorzata
M
Katedra Biochemii i Biotechnologii, Akademia Rolnicza, ul Wołyńska 35, 60-637 Poznań, Poland.
Stuible
Hans-Peter
HP
Kombrink
Erich
E
Guranowski
Andrzej
A
D000227:Adenine Nucleotides / Q000096:biosynthesis* / Q000378:metabolism; D017360:Arabidopsis / Q000201:enzymology* / Q000235:genetics; D002384:Catalysis; D003066:Coenzyme A Ligases / Q000378:metabolism*; D015226:Dinucleoside Phosphates / Q000096:biosynthesis; D007527:Isoenzymes / Q000378:metabolism; D008958:Models, Molecular; D009154:Mutation
D000227:Adenine Nucleotides; D015226:Dinucleoside Phosphates; D007527:Isoenzymes; C017991:adenosine 5'-tetraphosphate; C048567:adenosine 5'-pentaphosphate; D003066:Coenzyme A Ligases; C019943:4-coumarate-CoA ligase