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

**Chun-Yen Hsieh <sup>1</sup> , Yi-Hao Huang <sup>2</sup> , Hui-Hsuan Yeh <sup>2</sup> , Pei-Yu Hong <sup>2</sup> , Che-Jen Hsiao <sup>3</sup> and Lu-Sheng Hsieh 2,\***


**Abstract:** 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 ◦C, respectively. The *K*m values of BoPAL4 for three substrates were 2084, 98, and 956 µM, respectively. The *k*cat values of BoPAL4 for three substrates were 1.44, 0.18, and 0.06 σ −1 , respectively. The major substrate specificity site mutant, BoPAL4-H123F, showed better affinity toward L-phenylalanine by decreasing its *K*<sup>m</sup> value to 640 µM and increasing its *k*cat value to 1.87 s−<sup>1</sup> . 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.

**Keywords:** *Bambusa oldhamii*; phenylalanine ammonia-lyase; phenylalanine/tyrosine ammonia-lyase; substrate specificity; plant secondary metabolism; phenylpropanoid
