Molecular characterization of phenylalanine ammonia lyase gene from Cistanche deserticola.
Mol Biol Rep
; 38(6): 3741-50, 2011 Aug.
Article
en En
| MEDLINE
| ID: mdl-21104014
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.
Texto completo:
1
Colección:
01-internacional
Banco de datos:
MEDLINE
Asunto principal:
Fenilanina Amoníaco-Liasa
/
Genes de Plantas
/
Cistanche
Idioma:
En
Revista:
Mol Biol Rep
Año:
2011
Tipo del documento:
Article