Molecular characterization of the phenylalanine ammonia-lyase from Ephedra sinica.
The cDNAs (Espals) encoding phenylalanine ammonia-lyase (PAL) were cloned from Ephedra sinica by reverse transcription polymerase chain reaction (RT-PCR) using degenerate primers and by 5' and 3'-rapid amplification of cDNA ends (RACE). 2166 bp of the open reading frame (ORF) encoded 722 amino acids; sequence analyses of Espal clones suggested that at least four isoforms of EsPAL (EsPAL1, 2, 3, 4) existed, with nine amino acids substitution in their sequences. Phylogenetic analysis of EsPAL and PALs from other plant species revealed that EsPAL and Pinus PAL formed a gymnosperm-type PAL subfamily. The recombinant EsPAL1 to 4 functionally catalyzed a PAL reaction and their K(m), V(max), K(cat) and K(cat)/K(m) values did not show significant differences. Semi-quantitative RT-PCR analysis indicated that the expression of Espal genes in the roots was higher than in the plant's aerial parts. In addition, the activity of PAL in the roots was also higher than in the aerial parts. These results suggest that Espal genes are expressed in the whole plant but are dominant in the roots rather than in the aerial parts.
Okada
Taketo
T
Faculty of Pharmaceutical Sciences at Kagawa Campus, Tokushima Bunri University, Sanuki-city, Kagawa 769-2193, Japan. okadat@kph.bunri-u.ac.jp
Mikage
Masayuki
M
Sekita
Setsuko
S
D000595:Amino Acid Sequence; D015139:Blotting, Southern; D002851:Chromatography, High Pressure Liquid; D002934:Cinnamates / Q000737:chemistry / Q000378:metabolism; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000096:biosynthesis / Q000235:genetics; D018744:DNA, Plant / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D004591:Electrophoresis, Polyacrylamide Gel; D029789:Ephedra / Q000201:enzymology*; D008969:Molecular Sequence Data; D010650:Phenylalanine Ammonia-Lyase / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics*; D010802:Phylogeny; D018515:Plant Leaves / Q000737:chemistry; D018517:Plant Roots / Q000737:chemistry; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry; D020133:Reverse Transcriptase Polymerase Chain Reaction
D002934:Cinnamates; D018076:DNA, Complementary; D018744:DNA, Plant; D011994:Recombinant Proteins; D010650:Phenylalanine Ammonia-Lyase