Journal
ANALYTICAL BIOCHEMISTRY
Volume 302, Issue 2, Pages 305-312Publisher
ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1006/abio.2001.5574
Keywords
coenzyme A; electrospray ionizationmass spectrometry; affinity chromatography; solid-phase extraction; 4-coumarate; CoA ligase; benzoate; CoA ligase; recombinant proteins
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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 -80degreesC, thus providing a reliable and facile method to produce these delicate biological intermediates. (C) 2002 Elsevier Science (USA).
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