A Study On Active Center Of D-aminoacylase From M.natoriense TNJL143-2

D-aminoacylase has the ability to specifically catalyze the hydrolysis of N-acyl D-amino acid acyl groups.D-aminoacylase(AcyM)from Microbacterium natoriense TNJL143-2(M.natoriense TNJL143-2)has 24-27% homology with several known D-aminoacylases.Structural comparative analysis revealed that several known amino acid residues of the D-aminoacylase catalytically active center are also present in AcyM.In order to determine the active center of AcyM,we explored the relationship between AcyM structure and function.In this study,we used the enzyme encoding AcyM in M.natoriense TNJL143-2 to express the gene in Escherichia coli JM109(AcyM-pUC19).The partially conserved histidine residue and the 86 th alanine residue in the amino acid sequence of AcyM-pUC19 were replaced by site-directed mutagenesis,and the activities of various mutant enzymes were studied separately.The results of expression identification showed that each mutant enzyme was a soluble protein,and the activity identification showed that the activity of the mutant enzyme H61 A was 6% of the activity of AcyM-pUC19.The mutant enzymes H61 D,H63A,and H63 D were completely inactivated.The activities of the mutant enzymes H201 A and H201 D were approximately the same as those of AcyM-pUC19.The activities of the mutant enzymes H232 A and H232 D were 51% and 49%,respectively,of AcyM-pUC19 activity.The activity of the mutant enzyme A86 C is approximately the same as that of AcyM-pUC19.These results indicate that the selected mutation site contains key amino acid residues that affect enzyme activity,and in the spatial structure of the protein,these key amino acid residues may be located around the active center.The following hypothesis can be made by the change in the activity of each mutant enzyme: in the D-amino acylase derived from M.natoriense TNJL143-2,the 61 st amino acid sequence in the amino acid sequence is essential for catalysis.However,it is not necessary for substrate binding,and the 63 th histidine residue plays a decisive role in substrate binding.The 201 th histidine residue is not involved in catalysis and substrate recognition,and the 232 th histidine residue may catalyze and bind to the substrate.The aspartic acid residue that replaces the 86 th alanine residue is not involved in catalysis and substrate binding.