UBC Theses and Dissertations
Plant screening for tyrosine/phenylalanine ammonia lyase and biochemical characterisation, purification and cloning of the tyrosine/phenylalanine ammonia lyase enzyme from Trichosporon cutaneum Goldson, Andrea Michelle
Phenylalanine ammonia lyase (PAL, EC 18.104.22.168) catalyzes the conversion of phenylalanine (Phe) to trans cinnamic acid and, in some instances tyrosine (Tyr) to para hydroxycinnamic acid (pHCA). The specificity of PAL for Phe relative to Tyr varies by over 10⁶ between biological sources. An understanding of the basis for this astounding range of substrate preference is required to rationally engineer a highly efficient Tyr-specific enzyme for use in synthesizing pHCA for health, pharmaceutical and flavour applications. Of the plant seedlings screened for PAL activity, Triticum aestivum displayed the highest level of activity with both substrates. A unique 103 kDa PAL polypeptide was detected by Western blot analysis, along with two others at 74 and 83 kDa. Dual substrate activity was identified for the first time in the dicot Lens culinaris. Microbial Trichosporon cutaneum PAL (TcPAL) possessed the highest level of activity with Tyr of the sources investigated. Induction with Tyr (2 mM) produced the highest ratio of Phe:Tyr activity (1.6 ± 0.3: 0.4 ± 0.1 µmol/h g wet weight). TcPAL displayed Michaelis Menten kinetics with Phe (Km 5.0 ± 0.7 mM) but allosteric kinetics with Tyr (K' 1.7 ± 0.8 mM, Hill coefficient 1.8 ± 0.2). A greater specificity for Phe was demonstrated by a Phe [Vmax/Km] / Tyr [Vmax/Km] ratio of 2. The enzyme had a pH optimum of 8 - 8.5, temperature optimum of 32°C, showed no metal dependence, and had a monomer molecular mass of 79 kDa. Of the three methods investigated for purifying TcPAL, anion exchange chromatography using a Hi-Trap Q-Sepharose column produced the highest yield (20%) and purification fold (50). The PAL gene from Trichosporon cutaneum was cloned into the pET30a vector and sequenced. Five amino acid residues different from a previous report were identified, namely Arg74Glu, Val274Ala, Ala298Val, Ser322Pro and Arg486Lys. The gene had an intron of 1062 base pairs, starting at position 121. A His-Gln motif, which appears to be characteristic of yeast PALs with dual substrate activity, was identified in the substrate selectivity region. Residues that could potentially enhance the tyrosine activity of the enzyme were identified for future mutagenesis studies.
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