Transcriptome-wide identification and characterization of Ornithogalum saundersiae phenylalanine ammonia lyase gene family

Abstract

OSW-1 is a promising antitumor glycoside present in the Ornithogalum saundersiae plant. Biosynthesis of the p-methoxybenzoyl group on the disaccharide moiety of OSW-1 is known to take place biochemically by phenylpropanoid biosynthetic pathway, but molecular biological characterization of related genes has been insufficient. Phenylalanine ammonia lyase (PAL, EC 4.3.1.24), which catalyzes the deamination of L-phenylalanine to yield trans-cinnamic acid, plays a key role in phenylpropanoid metabolism. Thus, the study on the characterization of the genes involved in the OSW-1 biosynthetic pathway, particularly the well-documented genes such as PAL, is essential to further the understanding of the biosynthesis of OSW-1. Here, transcriptomic sequencing of O. saundersiae was performed to speed up the identification of a large number of genes related to OSW-1 biosynthesis. De novo assembly of the transcriptome sequence provided 210 733 contigs, 104, 180 unigenes, and four unigenes showing high similarities with PALs. Two full-length cDNAs encoding PALs (OsaPAL2 and OsaPAL62) from O. saundersiae were cloned using sequence information from these four unigenes. The PAL and tyrosine ammonia lyase (TAL) activities of recombinant OsaPAL proteins were unambiguously determined by HPLC with UV and MS detection as well as by NMR spectroscopy. Subsequently, a series of site-directed mutants were generated with the aim of improving enzyme activity and investigating the importance of particular residues in determining substrate selectivity. The results reveal that the Phe-to-His mutants, i.e., OsaPAL2F134H and OsaPAL62F128H, exhibited higher TAL activity than the corresponding wild types, providing direct evidence that the Phe residue is responsible for substrate specificity. Mutagenesis studies also demonstrated that the Thr-to-Ser mutants, i.e., OsaPAL2T196S and OsaPAL62T194S, showed significantly higher substrate affinity than the wild types. Furthermore, the Gly-to-Ala mutants, i.e., OsaPAL2G209A and OsaPAL62G207A, showed higher PAL and TAL activities. These findings provide further insight into the genes responsible for OSW-1 biosynthesis and will facilitate the future application of OsaPALs in synthetic biology.