Phenylalanine, Tyrosine, and DOPA Are bona fide Substrates for Bambusa oldhamii BoPAL4

Phenylalanine ammonia-lyase (PAL) links the plant primary and secondary metabolisms, and its product, trans-cinnamic acid, is derived into thousands of diverse phenylpropanoids. Bambusa oldhamii BoPAL4 has broad substrate specificity using L-phenylalanine, L-tyrosine, and L-3,4-dihydroxy phenylalanine (L-DOPA) as substrates to yield trans-cinnamic acid, p-coumaric acid, and caffeic acid, respectively. The optimum reaction pH of BoPAL4 for three substrates was measured at 9.0, 8.5, and 9.0, respectively. The optimum reaction temperatures of BoPAL4 for three substrates were obtained at 50, 60, and 40 & DEG;C, respectively. The Km values of BoPAL4 for three substrates were 2084, 98, and 956 mu M, respectively. The k(cat) values of BoPAL4 for three substrates were 1.44, 0.18, and 0.06 sigma(-1), respectively. The major substrate specificity site mutant, BoPAL4-H123F, showed better affinity toward L-phenylalanine by decreasing its K-m value to 640 mu M and increasing its k(cat) value to 1.87 s(-1). In comparison to wild-type BoPAL4, the specific activities of BoPAL4-H123F using L-tyrosine and L-DOPA as substrates retained 5.4% and 17.8% residual activities. Therefore, L-phenylalanine, L-tyrosine, and L-DOPA are bona fide substrates for BoPAL4.

Automated summary

Bambusa oldhamii BoPAL4 exhibits broad substrate specificity, with optimal reaction pH and temperature varying for different substrates. A major substrate specificity site mutant, BoPAL4-H123F, shows improved affinity for L-phenylalanine. BoPAL4 is confirmed to utilize L-phenylalanine, L-tyrosine, and L-DOPA as substrates.