Biosynthesis of 2-aminooctanoic acid and its use to terminally modify a lactoferricin B peptide derivative for improved antimicrobial activity

Abstract

Terminal modification of peptides is frequently used to improve their hydrophobicity. While N-terminal modification with fatty acids (lipidation) has been reported previously, C-terminal lipidation is limited as it requires the use of linkers. Here we report the use of a biocatalyst for the production of an unnatural fatty amino acid, (S)-2-aminooctanoic acid (2-AOA) with enantiomeric excess > 98% ee and the subsequent use of 2-AOA to modify and improve the activity of an antimicrobial peptide. A transaminase originating from Chromobacterium violaceum was employed with a conversion efficiency 52-80% depending on the ratio of amino group donor to acceptor. 2-AOA is a fatty acid with amino functionality, which allowed direct C- and N-terminal conjugation respectively to an antimicrobial peptide (AMP) derived from lactoferricin B. The antibacterial activity of the modified peptides was improved by up to 16-fold. Furthermore, minimal inhibitory concentrations (MIC) of C-terminally modified peptide were always lower than N-terminally conjugated peptides. The C-terminally modified peptide exhibited MIC values of 25 μg/ml for Escherichia coli, 50 μg/ml for Bacillus subtilis, 100 μg/ml for Salmonella typhimurium, 200 μg/ml for Pseudomonas aeruginosa and 400 μg/ml for Staphylococcus aureus. The C-terminally modified peptide was the only peptide tested that showed complete inhibition of growth of S. aureus.

Single point mutations reveal amino acid residues important for Chromobacterium violaceum transaminase activity in the production of unnatural amino acids

“…2-AOA (2-aminocaprylic acid) is used in the preparation of 1, 5-disubstituted-2-aminoimidazoles, which have antibiotic activity 34 . Furthermore, 2-AOA can be used to design a vaccine delivery system without the need for additional adjuvants 35 and to modify antimicrobial peptides for the improved activity 7 . 3-aminobutyric acid (3-ABA) or β-homoalanine has been used in the synthesis of a bioactive peptide for the treatment of autoimmune diseases 36 .…”

“…The transaminase gene of C . violaceum was amplified and cloned into pET-45b(+) vector as previously described 7 . Site-directed mutagenesis was performed using the QuickChange II XL Site-Directed Mutagenesis kit (Agilent Technologies, USA) according to manufacturer’s instructions.…”

“…The use of transaminases (TAs) in manufacturing optically pure chiral amines has been identified as one of the key emerging areas for the pharmaceutical industry 1 – 3 . Unnatural amino acids (UAAs) as chiral amines can be applied in the manufacture of diverse anticancer agents 4 – 6 , antimicrobials 7 , agrochemicals and other value added products 6 , 8 . In addition to their prominent role in peptide research and drug discovery, UAAs can be used to investigate the structure and dynamics of proteins, to study protein interactions, or to control the activity of proteins in living cells 9 .…”

“…Great interest in UAAs has inspired development of numerous methods for their synthesis, including biocatalysis. While the biocatalytic potential of TAs in the synthesis of UAAs has been demonstrated 7 , 10 , 11 , there is a growing need to expand the variety of UAAs which could be achieved by engineering existing TAs. While some TAs exhibit a naturally wide substrate range, the activity towards non-native substrates is usually tens of folds lower compared to the native substrate 12 .…”

“…We have previously shown that CV_TA has activity towards aliphatic 2-oxooctanoic acid (2-OOA) producing 2-aminooctanoic acid which can be used to make anti-bacterial peptides 7 . In the current study, using the reported CV_TA crystal structure 17 , 18 and the modelled relationship of CV_TA with 2-OOA as the amino group acceptor, specific amino acid residues were selected as targets for site directed mutagenesis.…”