Molecular characterization of a phenylalanine ammonia-lyase gene (BoPAL1) from Bambusa oldhamii.
Phenylalanine ammonia-lyase is the first enzyme of general phenylpropanoid pathway. A PAL gene, designated as BoPAL1, was cloned from a Bambusa oldhamii cDNA library. The open reading frame of BoPAL1 was 2,139 bp in size and predicted to encode a 712-amino acid polypeptide. BoPAL1 was the first intronless PAL gene found in angiosperm plant. Several putative cis-acting elements such as P box, GT-1motif, and SOLIPs involved in light responsiveness were found in the 5'-flanking sequence of BoPAL1 which was obtained by TAIL-PCR method. Recombinant BoPAL1 protein expressed in Pichia pastoris was active. The optimum temperature and pH for BoPAL1 activity was 50°C and 9.0, respectively. The molecular mass of recombinant BoPAL1 was estimated as 323 kDa using gel filtration chromatography and the molecular mass of full-length BoPAL was about 80 kDa, indicating that BoPAL1 presents as a homotetramer. The Km and kcat values of BoPAL1 for L-Phe were 1.01 mM and 10.11 s(-1), respectively. The recombinant protein had similar biochemical properties with PALs reported in other plants.
Hsieh
Lu-Sheng
LS
Institute of Microbiology and Biochemistry, Department of Biochemical Science and Technology, College of Life Science, National Taiwan University, 1 Roosevelt Road Section 4, Taipei, 106, Taiwan, ROC.
Hsieh
Yi-Lin
YL
Yeh
Chuan-Shan
CS
Cheng
Chieh-Yang
CY
Yang
Chien-Chih
CC
Lee
Ping-Du
PD
D024506:5' Flanking Region / Q000235:genetics; D031723:Bambusa / Q000201:enzymology* / Q000235:genetics*; D001483:Base Sequence; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D017343:Genes, Plant / Q000235:genetics*; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010650:Phenylalanine Ammonia-Lyase / Q000737:chemistry / Q000235:genetics*; D010843:Pichia / Q000378:metabolism; D010940:Plant Proteins / Q000737:chemistry / Q000235:genetics*; D011994:Recombinant Proteins / Q000302:isolation & purification; D012045:Regulatory Sequences, Nucleic Acid / Q000235:genetics; D013045:Species Specificity
D010940:Plant Proteins; D011994:Recombinant Proteins; D010650:Phenylalanine Ammonia-Lyase