Purification and characterization of prephenate aminotransferase from Anchusa officinalis cell cultures.

Prephenate aminotransferase (PAT) from rosmarinic acid-producing cell cultures of Anchusa officinalis has been purified to apparent electrophoretic homogeneity using a combination of high-performance anion-exchange, chromatofocusing, and gel filtration chromatography. The purified enzyme has a native molecular weight of 220,000 and subunit molecular weights of 44,000 and 57,000, indicating a possible alpha 2 beta 2 subunit structure. The purified PAT displays high affinity for prephenate (Km = 80 microM) but could also utilize other aromatic alpha-keto acids at less than 20% the rate with prephenate. L-Aspartate (Km = 80 microM) is about three times as effective as L-glutamate as amino-donor substrate. Anchusa PAT is not subject to feedback inhibition from L-phenylalanine or tyrosine, but its activity is affected by a rosmarinic acid metabolite, 3,4-dihydroxyphenyllactic acid.