Enzymatic synthesis and purification of aromatic coenzyme a esters.
Two recombinant His-tagged proteins, a plant 4-coumarate:coenzyme A ligase (EC 6.2.1.12) and a bacterial benzoate:coenzyme A ligase (EC 6.2.1.25), were expressed in Escherichia coli and purified in a single step using Ni-chelating chromatography. Purified enzymes were used to synthesize cinnamoyl-coenzyme A (CoA), p-coumaroyl-CoA, feruloyl-CoA, caffeoyl-CoA, and benzoyl-CoA. Conversions up to 95% were achieved. Using a rapid solid-phase extraction procedure, the target CoA esters were isolated with yields of up to 80%. Structures were confirmed by analytical comparison with chemically synthesized reference compounds and electrospray ionization-mass spectrometry. The recombinant enzymes were stable for several months at -80 degrees C, thus providing a reliable and facile method to produce these delicate biological intermediates.
Beuerle
Till
T
Department of Molecular, Cellular and Developmental Biology, University of Michigan, 830 North University Street, Ann Arbor, Michigan 48109-1048, USA.
Pichersky
Eran
E
D000214:Acyl Coenzyme A / Q000096:biosynthesis* / Q000302:isolation & purification; D002846:Chromatography, Affinity / Q000379:methods; D003066:Coenzyme A Ligases / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D010944:Plants / Q000201:enzymology; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism; D012015:Reference Standards; D012241:Rhodopseudomonas / Q000201:enzymology; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods
D000214:Acyl Coenzyme A; D011994:Recombinant Proteins; C119167:feruloyl-CoA; C058644:4-coumaroyl-coenzyme A; C058645:caffeoyl-coenzyme A; C037817:benzoyl-coenzyme A; C022903:cinnamoyl-coenzyme A; D003066:Coenzyme A Ligases; C019943:4-coumarate-CoA ligase; C055455:benzoate coenzyme A ligase