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.

Highly Active and Specific Tyrosine Ammonia-Lyases from Diverse Origins Enable Enhanced Production of Aromatic Compounds in Bacteria and Saccharomyces cerevisiae

“…The high pH optimum of the reactions may be a reflection of the reaction mechanism and is generally identical for all enzymes and similar to what has been observed previously for RsTAL, SeSam8, and other homologous enzymes (18,20,21,23,27,(78)(79)(80)(81). Alkaline pH may also be used for biocatalysis, where tyrosine produced by fermentation at physiological pH can be converted to pHCA in high titers by subsequent reaction at alkaline pH in the presence of TAL-expressing E. coli cells or cell paste (82).…”

Molecular Characterization of a Recombinant Zea mays Phenylalanine Ammonia-Lyase (ZmPAL2) and Its Application in trans-Cinnamic Acid Production from l-Phenylalanine

“…Thus, the optimal temperature of ZmPAL2 was 55°C. It was higher than that in R. chinensis and R. glutinis [17,23], similar to the ZmPAL1 and CdPAL from C. deserticola [29], and lower than that in S. miltiorrhiza [14]. As for pH, the pH profiles for the purified ZmPAL2 were shown in Fig.…”

“…Moreover, it was known that residue F137 in PALs played an important role in the substrate selectivity of PALs [44]. Those PALs that had TAL activity often had a histidine rather than a phenylalanine residue in the corresponding position [29]. According to the multiple alignments, AtPAL2, BoPAL1, and GbPAL had a phenylalanine, which did not effectively convert L-tyr to p-coumaric acid [10,15,18].…”

In vivo evolution of metabolic pathways: Assembling old parts to build novel and functional structures

“…Additionally Cd PAL is heat shock resistant: the protein retains 87% of its activity after heating (20 minutes at 70 C). 42 When both genes were subjected to in vivo evolution in DNA repair deficient cells, libraries were generated and clones were screened in their sensibility and/or resistance after heating and to use both Phe and Tyr as substrates. Proceeding in the same manner as with FLS recombinants, amid the PAL/TAL clones analyzed one of them with a single crossover showed a strong resistance to high temperatures and both, TAL and PAL activities were retained (Fig.…”