Abstract
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.
Highlights
The aromatic amino acid Phe, formed in both plants and microorganisms, serves as a building block for proteins and as a pathway intermediate to a wide range of aromatic compounds
Microbial prephenate dehydratase (PDT) are often allosterically regulated by binding of pathway end products [19], this being envisaged to occur through a C-terminal ACT domain, whereas cyclohexadienyl dehydratase (CDT) are not [18]
In Silico Analysis of Putative PDT and ACT Domains in Bacterial and Plant Genes and Proteins— arogenate dehydratase (ADT) activity has been biochemically detected in bacteria, plants, and green algae [16, 22, 23, 37, 38], there are no reports of molecular studies of any putative ADT genes in any organisms, only that of PDTs and CDTs
Summary
The aromatic amino acid Phe, formed in both plants and microorganisms, serves as a building block for proteins and as a pathway intermediate to a wide range of aromatic compounds. In Silico Analysis of Putative PDT and ACT Domains in Bacterial and Plant Genes and Proteins— ADT activity has been biochemically detected in bacteria, plants, and green algae [16, 22, 23, 37, 38], there are no reports of molecular studies of any putative ADT genes in any organisms, only that of PDTs and CDTs. Our in silico screening, resulted in the provisional identification of six putative PDT domaincontaining genes in Arabidopsis
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