1. Structure-function relationships of tachyplesins and their analogues
S Iwanaga, T Muta, T Shigenaga, N Seki, K Kawano, T Katsu, S Kawabata Ciba Found Symp. 1994;186:160-74; discussion 174-5. doi: 10.1002/9780470514658.ch10.
Haemocytes of the horseshoe crab (Limulus) contain a new family of arthropodous peptide antibiotics, termed the tachyplesin family. These cationic peptides are composed of 17-18 amino acid residues with a C-terminal arginine alpha-amide. Tachyplesin I takes on a fairly rigid conformation constrained by two disulphide bridges and adopts a conformation consisting of an antiparallel beta-sheet connected by a beta-turn. Isopeptides of tachyplesin I with amino acid replacements, tachyplesins II and III, and polyphemusins I and II have also been found in the haemocytes of the South-East Asian species and Limulus polyphemus. These peptides are present in abundance in the small granules of the haemocytes and inhibit strongly the growth of not only Gram-negative and Gram-positive bacteria but also fungi such as Candida albicans. Tachyplesin exists in the prepro form consisting of 77 residues; this precursor is probably processed by intracellular proteases and an amidation enzyme before incorporation into the small granules of the haemocytes. We examined the mode of action of tachyplesin I on biomembranes, comparing it with that of gramicidin S. Tachyplesin caused an efflux of K+ from Staphylococcus aureus and Escherichia coli cells similar to that caused by gramicidin S. Another antimicrobial substance, anti-LPS factor, has been isolated from haemocytes.
2. Alteration of substrate specificity of aspartase by directed evolution
Yasuhisa Asano, Ikuo Kira, Kenzo Yokozeki Biomol Eng. 2005 Jun;22(1-3):95-101. doi: 10.1016/j.bioeng.2004.12.002.
Aspartase (l-aspartate ammonia-lyase, EC 4.3.1.1), which catalyzes the reversible deamination of l-aspartic acid to yield fumaric acid and ammonia, is highly selective towards l-aspartic acid. We screened for enzyme variants with altered substrate specificity by a directed evolution method. Random mutagenesis was performed on an Escherichia coli aspartase gene (aspA) by error-prone PCR to construct a mutant library. The mutant library was introduced to E. coli and the transformants were screened for production of fumaric acid-mono amide from l-aspartic acid-alpha-amide. Through the screening, one mutant, MA2100, catalyzing deamination of l-aspartic acid-alpha-amide was achieved. Gene analysis of the MA2100 mutant indicated that the mutated enzyme had a K327N mutation. The characteristics of the mutated enzyme were examined. The optimum pH values for the l-aspartic acid and l-aspartic acid-alpha-amide of the mutated enzyme were pH 8.5 and 6.0, respectively. The K(m) value and V(max) value for the l-aspartic acid of the mutated enzyme were 28.3 mM and 0.26 U/mg, respectively. The K(m) value and V(max) value for the l-aspartic acid-alpha-amide of the mutated enzyme were 1450 mM and 0.47 U/mg, respectively. This is the first report describing the alteration of the substrate specificity of aspartase, an industrially important enzyme.
3. Synthesis of antimicrobial peptoids
Paul R Hansen, Jens K Munk Methods Mol Biol. 2013;1047:151-9. doi: 10.1007/978-1-62703-544-6_11.
Peptoids (N-substituted glycines) are mimics of α-peptides in which the side chains are attached to the backbone N (α) -amide nitrogen instead of the C (α) -atom. Peptoids hold promise as therapeutics since they often retain the biological activity of the parent peptide and are stable to proteases. In recent years, peptoids have attracted attention as new potential antibiotics against multiresistant bacteria. Here we describe the submonomer solid-phase synthesis of an antimicrobial peptoid, H-Nmbn-Nlys-Nlys-Nnap-Nbut-Nmbn-Nlys-NH2.
