title: Phenylalanine biosynthesis in Arabidopsis thaliana. Identification and characterization of arogenate dehydratases.
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.
authors.affiliation: Institute of Biological Chemistry, Washington State University, Pullman, Washington 99164-6340, USA.
authors.forename: Man-Ho
authors.initials: MH
authors.lastname: Cho
authors.forename: Oliver R A
authors.initials: OR
authors.lastname: Corea
authors.forename: Hong
authors.initials: H
authors.lastname: Yang
authors.forename: Diana L
authors.initials: DL
authors.lastname: Bedgar
authors.forename: Dhrubojyoti D
authors.initials: DD
authors.lastname: Laskar
authors.forename: Aldwin M
authors.initials: AM
authors.lastname: Anterola
authors.forename: Frances Anne
authors.initials: FA
authors.lastname: Moog-Anterola
authors.forename: Rebecca L
authors.initials: RL
authors.lastname: Hood
authors.forename: Susanne E
authors.initials: SE
authors.lastname: Kohalmi
authors.forename: Mark A
authors.initials: MA
authors.lastname: Bernards
authors.forename: ChulHee
authors.initials: C
authors.lastname: Kang
authors.forename: Laurence B
authors.initials: LB
authors.lastname: Davin
authors.forename: Norman G
authors.initials: NG
authors.lastname: Lewis
mesh_terms: D000600:Amino Acids, Dicarboxylic / Q000737:chemistry / Q000378:metabolism; D017360:Arabidopsis / Q000201:enzymology* / Q000235:genetics; D029681:Arabidopsis Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D003001:Cloning, Molecular; D003509:Cyclohexanecarboxylic Acids / Q000737:chemistry / Q000378:metabolism; D053138:Cyclohexenes / Q000737:chemistry / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D015870:Gene Expression; D006801:Humans; D006836:Hydro-Lyases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D010649:Phenylalanine / Q000096:biosynthesis* / Q000737:chemistry; D010802:Phylogeny; D011302:Prephenate Dehydratase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017434:Protein Structure, Tertiary / Q000502:physiology; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity / Q000502:physiology; D014443:Tyrosine / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism
chemical_list: D000600:Amino Acids, Dicarboxylic; D029681:Arabidopsis Proteins; D003509:Cyclohexanecarboxylic Acids; D053138:Cyclohexenes; D011994:Recombinant Proteins; D014443:Tyrosine; D010649:Phenylalanine; C015478:pretyrosine; D006836:Hydro-Lyases; C022437:pretyrosine dehydratase; D011302:Prephenate Dehydratase; C005550:prephenic acid