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.
De-Eknamkul
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Department of Chemistry, Guelph-Waterloo Centre for Graduate Work in Chemistry, University of Guelph, Ontario, Canada.
Ellis
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D002478:Cells, Cultured; D002845:Chromatography / Q000379:methods; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D007525:Isoelectric Focusing; D007700:Kinetics; D008970:Molecular Weight; D010944:Plants / Q000201:enzymology*; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D000637:Transaminases / Q000302:isolation & purification*
D000637:Transaminases; C044954:prephenate aminotransferase