Structure-based approach to alter the substrate specificity of Bacillus subtilis aminopeptidase

Abstract

Aminopeptidases can selectively catalyze the cleavage of the N-terminal amino acid residues from peptides and proteins. Bacillus subtilis aminopeptidase (BSAP) is most active toward p-nitroanilides (pNAs) derivatives of Leu, Arg, and Lys. The BSAP with broad substrate specificity is expected to improve its application. Based on an analysis of the predicted structure of BSAP, four residues (Leu 370, Asn 385, Ile 387, and Val 396) located in the substrate binding region were selected for saturation mutagenesis. The hydrolytic activity toward different aminoacyl-pNAs of each mutant BSAP in the culture supernatant was measured. Although the mutations resulted in a decrease of hydrolytic activity toward Leu-pNA, N385L BSAP exhibited higher hydrolytic activities toward Lys-pNA (2.2-fold) and Ile-pNA (9.1-fold) than wild-type BSAP. Three mutant enzymes (I387A, I387C and I387S BSAPs) specially hydrolyzed Phe-pNA, which was undetectable in wild-type BSAP. Among these mutant BSAPs, N385L and I387A BSAPs were selected for further characterized and used for protein hydrolysis application. Both of N385L and I387A BSAPs showed higher hydrolysis efficiency than the wild-type BASP and a combination of the wild-type and N385L and I387A BSAPs exhibited the highest hydrolysis efficiency for protein hydrolysis. This study will greatly facilitate studies aimed on change the substrate specificity and our results obtained here should be useful for BSAP application in food industry.

Keywords: Bacillus subtilis; aminopeptidase; protein hydrolysis; saturation mutagenesis; substrate specificity.

Figure 1. The structure of BSAP obtained by homology modeling. (A) Overall structure of BSAP. The α helices and β sheets are shown in red and cyan, respectively. Bestatin is shown in purple as the substrate. The PA-domain and substrate binding region are shown by dashed pane and dashed circle. (B) Local model of mutation sites in BSAP. The residues (Leu370, Asn385, Ile387, and Val396) in direct contact with the side chain of bound substrate are shown in balls and sticks, in which the oxygen atoms, nitrogen atoms, carbon atoms, and hydrogen atoms are in red, blue, deep gray, and light gray, respectively. The purple sticks indicate the bound substrate (bestatin). The two zinc atoms are shown in the form of magnified red balls. The distances (Å) from the bound substrate to the mutation sites in BSAP are shown. (C) The surface of the substrate binding region around the substrate in BSAP. The bound substrate is indicated by purple sticks. The residues Asn385 and Ile387 are displayed in the form of cyan and blue “CPK (Corey-Pauling-Koltun),” respectively. The pocket formed by the substrate binding sites is indicated by arrow.

Figure 2. SDS-PAGE analysis and specific activity of the purified mutant and wild-type BSAPs toward several aminoacyl-pNAs. (A) SDS-PAGE analysis of the purified mutant and wild-type BSAPs. Samples (15 μg of protein) were loaded on a 12% gel. Lanes: M, molecular mass marker; 1, N385L BSAP; 2, N385W BSAP; 3, I387A BSAP; 4, I387C BSAP; 5, I387S BSAP; 6, wild-type BSAP. (B) Substrate specificities of the positive mutant (N385L, N385W, I387A, I387C and I387S) and wild-type BSAPs toward four aminoacyl-pNAs (Leu-pNA, Lys-pNA, Met-pNA and Phe-pNA). The highest hydrolytic activity toward each aminoacyl-pNA is indicated by black bar. The values are the representative of three independent experiments. In all cases, the standard deviation was less than 5% of the mean. WT, wild-type.

Figure 3. Effect of temperature on the stability of N385L (), I387A () and wild-type () BSAPs. (A) Thermostability of those three BSAPs. (B) Thermal inactivation of those three BSAPs. The results are the mean of three independent experiments.

Figure 4. CD spectra of N385L, I387A and wild-type BSAPs. The secondary structural change of the mutants was checked by CD spectra in the far UV region (200‒250 nm).