Influence of Tyr78-loop on phenylalanine ammonia-lyase from Anabaena variabilis

Abstract:

[Background] Enzymatic property of the MIO-dependent (methylidene-imidazol-5-one) enzyme is significantly affected by the flexibility of the special loop that includes the catalysis Tyr (Tyr78-loop). [Objective] The effect of Tyr78-loop on activity of phenylalanine ammonia-lyase (PAL) from Anabaena variabilis was investigated to improve PAL catalytic activity. [Methods] Tyr78-loop was genetically modified. Positive mutants with enhanced activity were selected, followed by characterization. [Results] The specific activities of S73N, E95V, E95K and S73N/E95K mutants were improved by 34%, 30%, 18% and 35%, respectively, compared with that of the wild type at 37 °C and pH 8.5. According to protein structure simulation, the amino acid sites Asn73, Val95 and Lys95 in the mutants S73N, E95V and E95K, which located at the junction of α-helix and Tyr78-loop, had fewer hydrogen bonds with the nearby amino acids. This would increase the flexibility of Tyr78-loop and result in enhancement of the enzyme activity. [Conclusion] Catalytic activity of AvPAL could be improved by increasing the flexibility of Tyr78-loop through mutation on the Ser73 and Glu95 sites.

Key words:phenylalanine ammonia-lyase, activity, site-directed mutagenesis, stability