Preliminary characterization of sequence-activity relationship of Pseudomonas Aeruginosa arylsulfatase

Abstract:

Objective：To investigate the preliminary sequence-activity relationship of Pseudomonas Aeruginosa arylsulfatase（PAAS） by site-directed mutagenesis and to recognize residues suitable for iterative-saturation-mutagenesis. Methods：Candidates of residues involved in catalysis were recognized by analysis of conformation of PAAS active site；mutants were generated by site-directed muta－genesis and characterized after recombinant expression and Ni2+-NTA affinity purification. Results：In comparison to the wild type，the mutagenesis of R55，M72，G138，D317 and K375 caused the significant decrease of catalytic efficacy；the mutagenesis of R55，D317 and K375 reduced substrate preference for 4-nitrophenylsulfate and/or thermostability，but the mutagenesis of M72 and G138 caused primarily the decrease of activity. Conclusion：M72 and G138 are suitable candidate sites for directed evolution by iterative-satura－tion-mutagenesis to improve mutant activity.