title: Molecular characterization of phenylalanine ammonia lyase gene from Cistanche deserticola.
abstract: We cloned the gene, CdPAL1, from Cistanche deserticola callus using RACE PCR with degenerate primers that were designed based on a multiple sequence alignment of known PAL genes from other plant species. The gene shows high homology to other known PAL genes registered in GenBank. The recombinant protein exhibited Michaelis-Menten kinetics with a Km of 0.1013 mM, Vmax of 4.858 μmol min(-1), Kcat of 3.36 S(-1), and Kcat/Km is 33,168 M(-1) S(-1). The enzyme had an optimal pH of 8.5 and an activation energy of 38.92 kJ mol(-1) when L-Phenylalanine was used as a substrate; L-tyrosine cannot be used as substrate for this protein. The optimal temperature was 55°C, and the thermal stability results showed that, after a treatment at 70°C for 20 min, the protein retained 87% activity, while a treatment at 75°C for 20 min resulted in a loss of over 85% of the enzyme activity. Treatment with heavy metal ions (Hg2+, Pb2+, and Zn2+) showed remarkable inhibitory effects. Among the intermediates from the lignin (cinnamyl alcohol, cinnamyl aldehyde, coniferyl aldehyde, coniferyl alcohol), phenylpropanoid (cinnamic acid, coumaric acid, caffeic acid, and chlorogenic acid) and phenylethanoid (tyrosol and salidroside) biosynthetic pathways, only cinnamic acid showed strong inhibitory effects against CdPAL1 activity with a Ki of 8 μM. Competitive inhibitor AIP exhibited potent inhibition with Ki=0.056 μM.
authors.affiliation: College of Life Science and Natural Resources, Dong-A University, Busan, 604-714, Korea.
authors.forename: Gao Sheng
authors.initials: GS
authors.lastname: Hu
authors.forename: Jing Ming
authors.initials: JM
authors.lastname: Jia
authors.forename: Yeon Jae
authors.initials: YJ
authors.lastname: Hur
authors.forename: Young Soo
authors.initials: YS
authors.lastname: Chung
authors.forename: Jai Heon
authors.initials: JH
authors.lastname: Lee
authors.forename: Dae Jin
authors.initials: DJ
authors.lastname: Yun
authors.forename: Woo Sik
authors.initials: WS
authors.lastname: Chung
authors.forename: Gi Hwan
authors.initials: GH
authors.lastname: Yi
authors.forename: Tae Ho
authors.initials: TH
authors.lastname: Kim
authors.forename: Doh Hoon
authors.initials: DH
authors.lastname: Kim
mesh_terms: D031671:Cistanche / Q000187:drug effects / Q000201:enzymology* / Q000235:genetics*; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability / Q000187:drug effects; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects; D018506:Gene Expression Regulation, Plant / Q000187:drug effects; D017343:Genes, Plant / Q000235:genetics*; D006863:Hydrogen-Ion Concentration / Q000187:drug effects; D007477:Ions; D007700:Kinetics; D019216:Metals, Heavy / Q000494:pharmacology; D008969:Molecular Sequence Data; D009928:Organ Specificity / Q000187:drug effects; D010455:Peptides / Q000737:chemistry; D010636:Phenols / Q000378:metabolism; D010650:Phenylalanine Ammonia-Lyase / Q000235:genetics* / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D016415:Sequence Alignment; D020539:Sequence Analysis, Protein; D013056:Spectrophotometry, Ultraviolet; D013696:Temperature
chemical_list: D007477:Ions; D019216:Metals, Heavy; D010455:Peptides; D010636:Phenols; D011994:Recombinant Proteins; D010650:Phenylalanine Ammonia-Lyase