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Abstract
Almost all natural proteins are composed exclusively of l-amino acids, and this chirality influences their properties, functions, and selectivity. Proteases can recognize proteins composed of l-amino acids but display lower selectivity for their stereoisomers, d-amino acids. Taking this as an advantage, d-amino acids can be used to develop polypeptides or biobased materials with higher biostability. Chemoenzymatic peptide synthesis is a technique that uses proteases as biocatalysts to synthesize polypeptides, and d-stereospecific proteases can be used to synthesize polypeptides incorporating d-amino acids. However, engineered proteases with modified catalytic activities are required to allow the incorporation of d-amino acids with increased efficiency. To understand the stereospecificity presented by proteases and their involvement in polymerization reactions, we studied d-aminopeptidase. This enzyme displays the ability to efficiently synthesize poly d-alanine-based peptides under mild conditions. To elucidate the mechanisms involved in the unique specificity of d-aminopeptidase, we performed quantum mechanics/molecular mechanics simulations of its polymerization reaction and determined the energy barriers presented by the chiral substrates. The enzyme faces higher activation barriers for the acylation and aminolysis reactions with the l-stereoisomer than with the d-substrate (10.7 and 17.7 kcal mol−1 higher, respectively). The simulation results suggest that changes in the interaction of the substrate with Asn155 influence the stereospecificity of the polymerization reaction.
Highlights
Of the 20 canonical proteinogenic amino acids, all except Gly have a chiral centre at the Ca backbone atom, resulting in the presence of L- and D-stereoisomers
We polymerized the peptides with DAP under mild conditions, namely, 1 M PBS buffer at pH 8.0 and 25 C in the absence of any organic solvent based on our previous procedure for other enzymes.[29,48]
The reactions were conducted using BocD-alanine and D-Ala-OEt in chloroform, and the residue was puri ed to afford a white powder in 60% yield
Summary
Of the 20 canonical proteinogenic amino acids, all except Gly have a chiral centre at the Ca backbone atom, resulting in the presence of L- and D-stereoisomers. The protonation state of the amino acid residues for the MD was set according to the environment and previous studies; for modelling the acylation reaction, Lys[65] was set as neutral, and nearby residue His[287] was kept neutral and 3-protonated.[35,51] All molecular dynamic simulations were performed using AMBER 16.52 The atoms in the protein were described using the AMBER ff14SB force eld,[53] Land D-Ala-OEt substrates were described with a GAFF,[54] and the system was solvated with TIP3P water molecules.[55] A time step of 2 fs along with the SHAKE algorithm was used,[56] while the particle-mesh Ewald (PME) method[57] was used to calculate longrange interactions.
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