Identification of bean hydroxycinnamoyl-CoA:tetrahydroxyhexanedioate hydroxycinnamoyl transferase (HHHT): use of transgenic alfalfa to determine acceptor substrate specificity.

Transgenic alfalfa ( Medicago sativa L.) provides a useful reverse genetics platform to elucidate acceptor substrate specificity for uncharacterized BAHD family hydroxycinnamoyl-CoA hydroxycinnamoyl transferases. Tissues of many plant species accumulate hydroxycinnamoyl derivatives, often esters, thought to serve in protection against biotic and abiotic stresses. In many cases, these specialized metabolites are produced by BAHD family hydroxycinnamoyl-CoA hydroxycinnamoyl transferases (HCTs). Bean (Phaseolus vulgaris) leaves contain both hydroxycinnamoyl-malate esters and an HCT activity capable of making them. In seeking to identify this HCT from bean, we identified a gene whose predicted protein showed a high degree of sequence similarity (75%) to the Trifolium pratense (red clover) enzyme that carries out this reaction. The encoded bean protein, however, failed to carry out the malate transfer reaction when expressed in Escherichia coli. Expression of the gene in alfalfa (Medicago sativa) resulted in accumulation of several new hydroxycinnamates not present in nontransformed alfalfa, many of which corresponded to phenolics present in bean. Using accurate mass and UV absorption spectral data, we identified the acceptor substrate for this HCT as tetrahydroxyhexanedioic acids and demonstrated this predicted transferase activity with the E. coli-expressed protein. This finding adds to the growing number of BAHD family HCTs that have been characterized with respect to substrate specificity. Such data, combined with primary sequence and protein structural data will allow for a better understanding of the structure/function relationships of these enzymes and may eventually aid the rational design of such enzymes for altered substrate specificities. Additionally, expression of HCTs of unknown substrate specificity in alfalfa and characterization of the resulting accumulated novel metabolites could be a useful approach to characterizing putative BAHD HCT enzymes.