Phenylpyruvic acid

L-phenylalanine, one of the best substrates for pvLAAD, was chosen to evaluate the catalytic activities of different pvLAAD truncations and mutants. The transformation of L-phenylalanine by pvLAADs was measured by detecting the production of phenylpyruvic acid (PPA), which has an absorption peak at 321 nm. In brief, L-phenylalanine was dissolved to the concentration 25 mM in 500 μL of reaction buffer (50 mM potassium phosphate, pH 7.5) with or without 2 mg/mL of E. coli membrane. The pvLAADs were added at the final concentration of 10 μM to start the reactions. Reactions were incubated at 25 °C, and at the three time points (1 min, 10 min and 60 min), aliquots of 100 μL of the reactions were transferred into a 96-wells plate containing 50 μL of 3 M NaOH. The absorption at 321 nm was measured using microplate reader (Flex Station 3, Molecular Devices). The reaction without adding pvLAAD enzyme was used as the blank control. For quantification, phenylpyruvic acid (Sigma-Aldrich) at different concentrations (100 μM, 500 μM, 1000 μM, 2500 μM, 5000 μM, 10000 μM) were used to calculate the standard curve.

The substrates SHIK, phenylpyruvic acid, ATP, KCl, MgCl₂, PEP, FAD, NADH, β-Mercaptoethanol, L-Glutamic acid, pyridoxal phosphate were purchased from Sigma (St. Louis, MO, USA). Prime Star Taq polymerase was purchased from Takara and the restriction enzymes and T4 DNA ligase were purchased from NEB. Ni-NTA agarose resin was supplied by GE Healthcare for His-tagged protein purification. Na₂HPO₄ and other organic solvents used for HPLC were purchased from Merck (German). Other chemicals used in this article otherwise demonstrated were purchased from Solarbio (Beijing, China).
All kits and markers used for the construction of clones were from Omega Bio-tek, Inc (USA).