There is much uncertainty as to whether plants use
arogenate,
phenylpyruvate, or both as obligatory intermediates in Phe biosynthesis, an essential dietary
amino acid for humans. This is because both
prephenate and
arogenate have been reported to undergo decarboxylative
dehydration in plants via the action of either
arogenate (ADT) or
prephenate (
PDT)
dehydratases; however, neither
enzyme(s) nor encoding gene(s) have been isolated and/or functionally characterized. An in silico data mining approach was thus undertaken to attempt to identify the
dehydratase(s) involved in Phe formation in Arabidopsis, based on sequence similarity of
PDT-like and ACT-like domains in bacteria. This data mining approach suggested that there are six
PDT-like homologues in Arabidopsis, whose phylogenetic analyses separated them into three distinct subgroups. All six genes were cloned and subsequently established to be expressed in all tissues examined. Each was then expressed as a Nus fusion
recombinant protein in Escherichia coli, with their substrate specificities measured in vitro. Three of the resulting
recombinant proteins, encoded by ADT1 (At1g11790), ADT2 (At3g07630), and ADT6 (At1g08250), more efficiently utilized
arogenate than
prephenate, whereas the remaining three, ADT3 (At2g27820), ADT4 (At3g44720), and ADT5 (At5g22630) essentially only employed
arogenate. ADT1, ADT2, and ADT6 had k(cat)/Km values of 1050, 7650, and 1560 M(-1) S(-1) for
arogenate versus 38, 240, and 16 M(-1) S(-1) for
prephenate, respectively. By contrast, the remaining three, ADT3, ADT4, and ADT5, had k(cat)/Km values of 1140, 490, and 620 M(-1) S(-1), with
prephenate not serving as a substrate unless excess
recombinant protein (>150 microg/assay) was used. All six genes, and their corresponding
proteins, are thus provisionally classified as
arogenate dehydratases and designated ADT1-ADT6.