Versatile Peptide C-Terminal Functionalization via a Computationally Engineered Peptide Amidase

Abstract

The properties of synthetic peptides, including potency, stability, and bioavailability, are strongly influenced by modification of the peptide chain termini. Unfortunately, generally applicable methods for selective and mild C-terminal peptide functionalization are lacking. In this work, we explored the peptide amidase from Stenotrophomonas maltophilia as a versatile catalyst for diverse carboxy-terminal peptide modification reactions. Because the scope of application of the enzyme is hampered by its mediocre stability, we used computational protein engineering supported by energy calculations and molecular dynamics simulations to discover a number of stabilizing mutations. Twelve mutations were combined to yield a highly thermostable (Δ T m = 23 °C) and solvent-compatible enzyme. Protein crystallography and molecular dynamics simulations revealed the biophysical effects of mutations contributing to the enhanced robustness. The resulting enzyme catalyzed the selective C-terminal modification of synthetic peptides with small nucleophiles such as ammonia, methylamine, and hydroxylamine in various organic (co)­solvents. The use of a nonaqueous environment allowed modification of peptide free acids with >85% product yield under thermodynamic control. On the basis of the crystal structure, further mutagenesis gave a biocatalyst that favors introduction of larger functional groups. Thus, the use of computational and rational protein design provided a tool for diverse enzymatic peptide modification.

Discovery of aPseudomonas aeruginosaType VI secretion system toxin targeting bacterial protein synthesis using a global genomics approach

“…Reactions were carried out in two different buffers to test the possible effect of pH; one set of reactions was carried out in 10 mM sodium phosphate buffer (pH 7.6) and another set of reactions was carried out in 20 mM Tris-HCl buffer (pH 8.3). For epinecidin-1, 5 μM of freshly-purified Tse8 or the positive control protein Pam (purified as described previously 50 ), were incubated with 50 μM of putative substrate in 10 mM sodium phosphate buffer (pH 7.2); control reactions, lacking Tse8 or Pam, were also tested. Reactions were incubated overnight at 30 °C, followed by MS analysis.…”

“…The antimicrobial peptide epinecidin-1, as well as sermorelin, have amidated C termini. The latter has previously been used to measure the amidase activity of Pam from S. maltophilia 50 . Top panel in both (a) and (b) : Sequence covered by the fragments obtained after fragmentation of epinecidin-1 in the MS is indicated above the fragmentation spectra for epinecidin-1.…”

Crystal structure of a lipase from Streptomyces sp. strain W007 – implications for thermostability and regiospecificity

“…Fluorescence-based thermal shift assay was performed using a CFX connect Real-Time PCR instrument (Bio-Rad, Hercules, CA, USA) [44]. A sample of 20 lL of protein solution in buffer (20 mM phosphate buffer, pH 7.0) was mixed with 5 lL of 100-fold diluted Sypro Orange dye (Sigma-Aldrich, St Louis, MO, USA) in a thin-wall 96-well PCR plate.…”

Coevolution of both Thermostability and Activity of Polyphosphate Glucokinase from Thermobifida fusca YX

“…(ii) F30L and R34P mutations may result in a cooperative effect due to their hydrophobic interactions. The S␦ atom of M88 makes hydrophobic interactions with A43, P74, and W92 to fill in the deeply buried cavity surrounded by several loops (47). The substitution of alanine to threonine at position 231 stabilized the protein by introducing a new hydrogen bond with E202.…”