The Characterization And Mutational Analysis Of Ornithogalum Saundersiae Phenylalanine Ammonialyase Gene Family

OSW-1is a promising antitumor glycoside present in the plant Ornithogalum saundersiae. According to previous structure-activity relationship (SAR) studies, the p-hydroxybenzoic acid group is important for the cytotoxicity of OSW-1.Biosynthesis of this group is known to take place biochemically by phenylpropanoid biosynthetic pathway, but molecular biological characterization of related genes has been insufficient.To date, none of the Phenylalanine ammonia-lyase (PAL, EC4.3.1.24) in O. saundersiae has been cloned and characterized. PAL catalyzing the deamination of L-phenylalanine yielding trans-cinnamic acid, plays a key role in the ability of phenylpropanoid metabolism to channel carbon to produce p-methoxybenzoyl group on the disaccharide moiety of OSW-1. Thus, the study on the characterization of PAL, is essential to further understanding the biosynthesis of OSW-1.Functional analysis of wild-type recombinant OsaPAL proteinsFunctional analysis of the purified recombinant OsaPAL proteins was performed using L-Phe and L-Tyr as substrates and the corresponding PAL and tyrosine ammonialyase (TAL) activities unambiguously determined by HPLC with UV and MS detection, as well as by NMR spectroscopy. Biochemical analysis had also been performed.Results showed that both wild-type enzymes, OsaPAL2and OsaPAL62, could catalytic L-Tyr and L-Phe, and were demonstrated to have similar optimum pH and temperature.Pheâ†'His mutation significantly increased TAL activity of wild-type enzymesOsaPAL showed higher catalytic activity toward L-Phe than that toward L-Tyr, the low L-Tyr deamination activity would block its further application in metabolic engineering. To improve this activity, Phe134and Phe128of OsaPAL2and OsaPAL62, respectively, were substituted with His. TAL activity significantly improved, showing that this residue is associated with substrate specificity.Residue substitution successfully improves both PAL and TAL activityComparison of the PAL sequences of plants with those of yeasts revealed three residues differing around the active motif. These three residues were substituted with those found in yeast, resulting in the construction of three single mutants, one double mutant, and one triple mutant of OsaPAL2and OsaPAL62. The results showed that Thrâ†'Ser mutants (OsaPAL2T196S and OsaPAL62T194S) could significantly increase enzyme affinity to substrates and that Glyâ†'Ala mutants (OsaPAL2G209A and OsaPAL62G207A) could improve PAL and TAL activity. These results indicate that these three residues have different effects on the enzymes and provide a new way to improve their native activity.These genetic and biochemical findings provide the first insight into the genes responsible for OSW-1biosynthesis and will facilitate the future application of OsaPALs in synthetic biology.