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 K (m) of 0.1013 mM, V (max) of 4.858 mu mol min(-1), K (cat) of 3.36 S-1, and K (cat)/K (m) 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 55A degrees C, and the thermal stability results showed that, after a treatment at 70A degrees C for 20 min, the protein retained 87% activity, while a treatment at 75A degrees 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 K (i) of 8 mu M. Competitive inhibitor AIP exhibited potent inhibition with K (i) = 0.056 mu M.