Trichosporon cutaneum phenylalanine ammonia lyase was selected as a model to investigate the dual substrate activity of this family of enzymes. Sequencing of the PAL gene identified an extensive intron region at the N-terminus. Five amino acid residues differing from a prior report were identified. Highest Phe？:？Tyr activities ( : ？μmol/h？g wet weight) were induced by Tyr. The enzyme has a temperature optimum of 32°C and a pH optimum of 8–8.5 and shows no metal cofactor dependence. Michaelis-Menten kinetics (Phe, ？mM) and positive allostery (Tyr, ？mM, Hill coefficient ) were observed. Anion exchange chromatography gave a purification fold of 50 with 20% yield. The His-Gln motif (substrate selectivity switch region) indicates the enzyme’s ability to act on both substrates. 1. Introduction Phenylalanine ammonia lyase (PAL, EC 4.3.1.24) catalyzes the conversion of phenylalanine to trans-cinnamic acid, as a step in the phenylpropanoid pathway of plants and in the formation of secondary products of metabolism in some microorganisms [1, 2]. In some instances, the enzyme also converts tyrosine to para-hydroxycinnamic acid. These dual substrate enzymes are classified as phenylalanine/tyrosine ammonia lyases, (EC 4.3.1.25). Enzymes with a greater catalytic efficiency for tyrosine are known as tyrosine ammonia lyase (TAL, EC 4.3.1.23). There are no known genes that code for a lyase that has activity exclusively with tyrosine. Interest in PAL is from two perspectives. First, the structural features of the enzyme responsible for its substrate specificity have not been fully elucidated. Second, a more selective and efficient TAL is of interest for industrial applications. The specificity of PAL for phenylalanine relative to tyrosine varies by over 106 between biological sources, and typically, the efficiency of Phe turnover is higher than Tyr [3, 4]. An understanding of the basis for this astounding range of substrate preference is required to rationally engineer an efficient tyrosine-specific enzyme for use in the synthesis of p-hydroxycinnamic acid for industrial applications [5]. The microbial production of aromatic chemicals continues to increase as it allows for the use of greener technologies and renewable energy sources [6]. PAL has been extensively characterized from a wide variety of plant sources [7], but only a few microbial sources of the enzyme have been investigated [8, 9]. PAL from the yeast Trichosporon cutaneum (TcPAL), identified as an enzyme able to metabolize both Phe and Tyr and possessing a relatively high level of activity with tyrosine, was
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