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Acylaminoacyl peptidase (APH) belongs to the prolyl oligopeptidase (POP) family of serine protease.The putative APH gene from thermophilic Aeropyrum pernix K1 has been cloned,expressed in E.coli.apAPH showed both esterase and peptidase activities.We have determined the crystal structure of apAPH[1].Sequence alignment suggested there is an obvious bias towards arginine at position 526 in POP family,whereas leucine is favored in lipase family.The role of Arg526 was investigated by saturation mutagenesis.The specific value of (kcat/Km)esterase/(kcat/Km)peptidase has been changed from ~7 for the wild type to ~150 for mutant R526V.The structural and energetic effects on kinetic parameters were discussed by molecular dynamics simulation.These results provide rare examples how enzymes can be evolved to discriminate their substrates by a single mutation,and provide the first evolutionarily related evidence between the POP and lipase families[2].