Amino Acids Lanthionine Research Articles

Cross-linking of wheat gluten proteins during production of hard pretzels

The impact of the hot alkaline dip, prior to pretzel-baking, on the types and levels of cross-links between wheat proteins was studied. Protein extractability of pretzel dough in sodium dodecyl sulfate containing buffer decreased during alkaline dipping [45s, 1.0% (w/v) NaOH, 90°C], and even more during baking (3min at 250°C) and drying (10min at 135°C). Reducing agent increased the extractability partly, indicating that both reducible (disulfide, SS) and non-reducible (non-SS) protein cross-links had been formed. The decrease in cystine levels suggested β-elimination of cystine releasing Cys and dehydroalanine (DHA). Subsequent reaction of DHA with Lys and Cys, induced the unusual and potentially cross-linking amino acids lysinoalanine (LAL) and lanthionine (LAN), respectively, in alkaline dipped dough (7μmol LAN/g protein) and in the end product (9μmol LAL and 50μmol LAN/g protein). The baking/drying step increased sample redness, decreased Lys levels more than expected based on LAL formation (57μmol/g protein), and induced a loss of reducing sugars (99μmol/g protein), which suggested the potential contribution of Maillard-derived cross-links to the observed extractability loss. However, levels of Maillard products which possibly cross-link proteins, are small compared to DHA-derived cross-links. Higher dipping temperatures, longer dipping times, and higher NaOH concentrations increased protein extractability losses and redness, as well as LAL and LAN levels in the end product. No indications for Maillard-derived cross-links or LAL in pretzel dough immediately after dipping were found, even when severe dipping conditions were used.

Expression of the Lantibiotic Mersacidin in Bacillus amyloliquefaciens FZB42

Lantibiotics are small peptide antibiotics that contain the characteristic thioether amino acids lanthionine and methyllanthionine. As ribosomally synthesized peptides, lantibiotics possess biosynthetic gene clusters which contain the structural gene (lanA) as well as the other genes which are involved in lantibiotic modification (lanM, lanB, lanC, lanP), regulation (lanR, lanK), export (lanT(P)) and immunity (lanEFG). The lantibiotic mersacidin is produced by Bacillus sp. HIL Y-85,54728, which is not naturally competent.Methodology/Principal FindingsThe aim of these studies was to test if the production of mersacidin could be transferred to a naturally competent Bacillus strain employing genomic DNA of the producer strain. Bacillus amyloliquefaciens FZB42 was chosen for these experiments because it already harbors the mersacidin immunity genes. After transfer of the biosynthetic part of the gene cluster by competence transformation, production of active mersacidin was obtained from a plasmid in trans. Furthermore, comparison of several DNA sequences and biochemical testing of B. amyloliquefaciens FZB42 and B. sp. HIL Y-85,54728 showed that the producer strain of mersacidin is a member of the species B. amyloliquefaciens.Conclusions/SignificanceThe lantibiotic mersacidin can be produced in B. amyloliquefaciens FZB42, which is closely related to the wild type producer strain of mersacidin. The new mersacidin producer strain enables us to use the full potential of the biosynthetic gene cluster for genetic manipulation and downstream modification approaches.

Hydrolysis of Animal Protein Meals for Improved Utility in Non-Feed Applications

Rendered proteins are well suited for animal nutrition applications, but due to their insolubility, inhomogeneity, and the presence of non-protein substances, they are difficult to utilize in other applications. In an attempt to overcome these obstacles to utilization, three types of rendered proteins [meat and bone meal (MBM), feather meal (FM), and blood meal (BM)] were partially defatted and then hydrolyzed to varying extents using calcium hydroxide or one of three enzymatic treatments, in 4- or 6- L batches. After centrifugation, filtration, and spray drying, these hydrolysates were analyzed for changes in physical and chemical properties that relate to their potential utility. In all cases, the proportion of organic matter solubilized increased along with hydrolysis duration, although the molar mass distribution of the hydrolysis product only had a weak dependence on hydrolysis duration; the soluble material consisted of very small peptides at all time points. Alkali-hydrolysis was not effective in yielding a product low in ash; although the insoluble ash in MBM and FM appears not to have been carried over into the product, it was replaced by significant amounts of calcium salts; corresponding enzymatically-hydrolyzed batches contained approximately 40% less ash. Alkali-hydrolysis in particular had effects on the amino acid composition of the products, destroying some amino acids and creating others, including the cross-linked amino acids lysinoalanine and lanthionine; enzymatic hydrolysis effects on amino acid composition were different in type and generally lesser in magnitude. It is concluded that hydrolysis is a promising treatment for increasing the non-feed utility of rendered animal proteins.

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Isolation and characterization of a new low-molecular antibacterial peptide of the lantibiotics family

The physicochemical and biological properties of the low-molecular antibacterial peptide isolated from the cultivation medium of Staphylococcus warneri IEGM KL-1 were studied. The peptide was obtained in a homogenous state by the methods of ultrafiltration, ion exchange, and reversed phase chromatography. The peptide contained a substantial quantity of cationic and hydrophobic amino acid residues and an uncommon amino acid lanthionine. The molecular mass of the peptide was 2999 Da. A bactericidal effect of the isolated peptide on the cells of S. epidermidis 33 was exhibited in a wide pH range, being completely preserved upon heat treatment. In accordance with the characteristics, origin, and species affiliation of the producer, the peptide was named warnerin. The available data allow us to consider warnerin as a new representative of the family of lantibiotics, promising antibiotic agents of microbial origin.

Production of the Novel Two-Peptide Lantibiotic Lichenicidin by Bacillus licheniformis DSM 13

BackgroundLantibiotics are small microbial peptide antibiotics that are characterized by the presence of the thioether amino acids lanthionine and methyllanthionine. Lantibiotics possess structural genes which encode inactive prepeptides. During maturation, the prepeptide undergoes posttranslational modifications including the introduction of rare amino acids as lanthionine and methyllanthione as well as the proteolytic removal of the leader. The structural gene (lanA) as well as the other genes which are involved in lantibiotic modification (lanM, lanB, lanC, lanP), regulation (lanR, lanK), export (lanT(P)) and immunity (lanEFG) are organized in biosynthetic gene clusters.Methodology/Principal FindingsSequence comparisons in the NCBI database showed that Bacillus licheniformis DSM 13 harbours a putative lantibiotic gene cluster which comprises two structural genes (licA1, licA2) and two modification enzymes (licM1, licM2) in addition to 10 ORFs that show sequence similarities to proteins involved in lantibiotic production. A heat labile antimicrobial activity was detected in the culture supernatant and a heat stabile activity was present in the isopropanol cell wash extract of this strain. In agar well diffusion assays both fractions exhibited slightly different activity spectra against Gram-positive bacteria. In order to demonstrate the connection between the lantibiotic gene cluster and one of the antibacterial activities, two Bacillus licheniformis DSM 13 mutant strains harbouring insertions in the structural genes of the modification enzymes licM1 and licM2 were constructed. These strains were characterized by a loss of activity in the isopropanol extract and substractive MALDI-TOF predicted masses of 3020.6 Da and 3250.6 Da for the active peptides.Conclusions/SignificanceIn conclusion, B. licheniformis DSM 13 produces an antimicrobial substance that represents the two-peptide lantibiotic lichenicidin and that shows activity against a wide range of Gram-positive bacteria including methicillin resistant Staphylococcus aureus strains.

Identification and characterization of a bioactive lantibiotic produced by Staphylococcus warneri

Lantibiotics are a group of potent antibacterial agents that contain unusual amino acids, such as the thioether amino acids lanthionine and methyllanthionine, and the didehydroamino acids didehydroalanine and didehydro-aminobutyric acid. Here, we report on an antibacterial lantibiotic peptide named SWLP1 (Staphylococcus warneri lantibiotic peptide 1), which is secreted from Staphylococcus warneri (deposited with DSMZ, accession number DSM 16081). SWLP1 was purified from growth media. The purified peptide displays antibacterial activity against several species, including Staphylococcus epidermidis. The molecular mass of SWLP1 is 2998.9 Da as determined by matrix-assisted laser desorption ionization time-of-flight mass spectrometry. The sequence and possible structure was elucidated by combining electrospray ionization mass spectrometry/mass spectrometry data of ethanethiol-treated and non-ethanethiol-treated tryptic fragments of the SWLP1. SWLP1 contains three thioether bridges, one didehydroalanine, and three didehydroaminobutyric acids. This peptide has the potential to be used in treatment of several Gram-positive bacterial infections.

Investigation of the substrate specificity of lacticin 481 synthetase by using nonproteinogenic amino acids.

Lantibiotics are peptide antimicrobial compounds that are characterized by the thioether-bridged amino acids lanthionine and methyllanthionine. For lacticin 481, these structures are installed in a two-step post-translational modification process by a bifunctional enzyme, lacticin 481 synthetase (LctM). LctM catalyzes the dehydration of Ser and Thr residues to generate dehydroalanine or dehydrobutyrine, respectively, and the subsequent intramolecular regio- and stereospecific Michael-type addition of cysteines onto the dehydroamino acids. In this study, semisynthetic substrates containing nonproteinogenic amino acids were prepared by expressed protein ligation and [3+2]-cycloaddition of azide and alkyne-functionalized peptides. LctM demonstrated broad substrate specificity toward substrates containing beta-amino acids, D-amino acids, and N-alkyl amino acids (peptoids) in certain regions of its peptide substrate. These findings showcase its promise for use in lantibiotic and peptide-engineering applications, whereby nonproteinogenic amino acids might impart improved stability or modulated biological activities. Furthermore, LctM permitted the incorporation of an alkyne-containing amino acid that can be utilized for the site-selective modification of mature lantibiotics and used in target identification.

Biosynthesis and Biological Activities of Lantibiotics with Unique Post-Translational Modifications

Lantibiotics are biologically active peptides which contain the thioether amino acid lanthionine as well as several other modified amino acids. They can be broadly divided into two groups on the basis of their structures: type-A lantibiotics are elongated, amphiphilic peptides, while type-B lantibiotics are compact and globular. In the last decade there has been a marked increase in research interest in these peptides due both to the novel biosynthetic mechanisms by which they are produced, as well as to their potential applications. Lantibiotics are synthesised on the ribosome as a prepeptide which undergoes several post-translational modification events, including dehydration of specific hydroxyl amino acids to form dehydroamino acids, addition of neighbouring sulfhydryl groups to form thioethers and, in specific cases, other modifications such as introduction of D-alanine residues from L-serine, formation of lysinoalanine bridges, formation of novel N-terminal blocking groups and oxidative decarboxylation of a C-terminal cysteine. The genetic elements responsible for these specific modification reactions encode unique enzymes with hitherto unknown reaction mechanisms. Production of these peptides also requires accessory proteins including processing proteases, translocators of the ATP-binding cassette transporter family, regulatory proteins and dedicated producer self-protection mechanisms. While the principle biological activity of most type-B lantibiotics appears to be directed at the inhibition of enzyme functions, the type-A lantibiotics kill bacterial cells by forming pores in the cytoplasmic membrane.

Discovery and in vitro biosynthesis of haloduracin, a two-component lantibiotic.

Lantibiotics are ribosomally synthesized peptides that undergo posttranslational modifications to their mature, antimicrobial form. They are characterized by the unique amino acids lanthionine and methyllanthionine, introduced by means of dehydration of Ser/Thr residues followed by reaction of the resulting dehydro amino acids with cysteines to form thioether linkages. Two-component lantibiotics use two peptides that are each posttranslationally modified to yield two functionally distinct products that act in synergy to provide bactericidal activity. By using genetic data instead of isolation, a two-component lantibiotic, haloduracin, was identified in the genome of the Gram-positive alkaliphilic bacterium Bacillus halodurans C-125. We show that heterologously expressed and purified precursor peptides HalA1 and HalA2 are processed by the purified modification enzymes HalM1 and HalM2 in an in vitro reconstitution of the biosynthesis of a two-component lantibiotic. The activity of each HalM enzyme is substrate-specific, and the assay products exhibit antimicrobial activity after removal of their leader sequences at an engineered Factor Xa cleavage site, indicating that correct thioether formation has occurred. Haloduracin's biological activity depends on the presence of both modified peptides. The structures of the two mature haloduracin peptides Halalpha and Halbeta were investigated, indicating that they have similarities as well as some distinct differences compared with other two-component lantibiotics.

Engineering Dehydro Amino Acids and Thioethers into Peptides Using Lacticin 481 Synthetase

Lantibiotics are peptide antimicrobials containing the thioether-bridged amino acids lanthionine (Lan) and methyllanthionine (MeLan) and often the dehydrated residues dehydroalanine (Dha) and dehydrobutyrine (Dhb). While biologically advantageous, the incorporation of these residues into peptides is synthetically daunting, and their production in vivo is limited to peptides containing proteinogenic amino acids. The lacticin 481 synthetase LctM offers versatile control over the installation of dehydro amino acids and thioether rings into peptides. In vitro processing of semisynthetic substrates unrelated to the prelacticin 481 peptide demonstrated the broad substrate tolerance of LctM. Furthermore, a chemoenzymatic strategy was employed to generate novel thioether linkages by cyclization of peptidic substrates containing the nonproteinogenic cysteine analogs homocysteine and beta-homocysteine. These findings are promising with respect to the utility of LctM toward preparation of conformationally constrained peptide therapeutics.

Biochemical Studies of Nisin Biosynthesis and Autoimmunity

Lantibiotics derive their name from the unusual amino acid lanthionine, which consists of two alanine residues cross-linked by a thioether bridge that connects their β-carbons. Although the presence of the lanthionine thioether bridge is the characteristic structural motif found in all lantibiotics, they may also contain dehydrated amino acids such as 2,3-didehydroalanine (Dha) and 2,3-didehydrobutyrine (Dhb) as well as a methyl-substituted lanthionine derivative (2S,3S,6R)-3-methyllanthionine. Lantibiotics possess high levels of antimicrobial activity against several pathogenic gram-positive bacteria such as staphylococci, streptococci, and clostridia, and nisin, the prototypical lantibiotic, has been used extensively as a commercial food preservative. Despite the widespread use of nisin as a food preservative and its presence since antiquity, nisin still possesses unfettered cytotoxicity against Gram-positive bacteria with no significant environmental resistance being demonstrated. The lantibiotic nisin is a 34-residue bacteriocin produced by the Gram-positive bacteria Lactococcus lactis spp where it is first ribosomally synthesized as an inactive precursor and then post-translationally modified into its mature biologically active form. Described herein is a two-part study investigating (1) the basis of how the characteristic nisin lanthionine rings are installed by the enzyme NisC and (2) how Lactococcus lactis spp. is able to demonstrate an autoimmunity to the toxic effects of nisin despite producing such an effective bacteriocin such as nisin. Funding: American Cancer Society, NIH NIDA Individual NRSA (1F30DA019363), and the University of Illinois Institute for Genomic Biology.

Production and purification of a bacteriocin peptide produced by Lactococcus sp. strain GM005, isolated from Miso-paste

Lactococcus sp. GM005 was isolated from Miso-paste and was found to produce a bacteriocin with strong antibacterial activity. A culture of Lactococcus sp. GM005, maintained at 30 °C and a constant pH of 6.0, exhibited bacteriocin activity eightfold higher than that of a culture grown under pH-uncontrolled conditions. GM005 bacteriocin was purified to homogeneity on SDS-PAGE by hydrophobic column chromatography and gel filtration. The estimated molecular weight of GM005 bacteriocin was approximately 9.6 kDa based on gel-filtration analysis, and was approximately 2.4 kDa based on tricine-SDS-PAGE analysis, indicating a tetrametric structure. N-terminal amino acid analysis revealed that the N-terminal end was blocked. Amino acid composition analysis revealed a high proportion of hydrophobic amino acid residues and lanthionine. This differs from the composition of some lantibiotics. GM005 bacteriocin exhibits a bactericidal activity against Lactobacillus sakei JCM1157 T.

Multiple activities in lantibiotics--models for the design of novel antibiotics?

Lantibiotics are antibiotic peptides distinguished by the presence of the rare thioether amino acids lanthionine and/or methyllanthionine. They are produced by Gram-positive bacteria as gene-encoded precursor peptides and undergo post-translational modification to generate the mature peptide. The structural gene for the prepeptide and the genes involved in biosynthesis, processing, export as well as regulation and producer strain self-protection are organized in clusters. Based on their structural and functional features lantibiotics are currently divided into two major groups. The flexible amphiphilic type-A lantibiotics act primarily by pore formation in the bacterial membrane, a mechanism which was recently shown, e.g. for nisin and epidermin, to involve the interaction with specific docking molecules such as the membrane precursor lipid II. The rather rigid and globular type-B lantibiotics inhibit enzyme functions through interaction with the respective substrates: mersacidin and actagardine inhibit the cell wall biosynthesis by complexing lipid II, whereas the cinnamycin-like peptides inhibit phospholipases by binding phosphoethanolamine. Lantibiotics have attracted much attention in recent years and undergone extensive characterization. New insights into the mode of action and structure-function relationships as well as the biochemistry and the genetics will be outlined in this review.

Posttranslationally modified bacteriocins—the lantibiotics

Lantibiotics are a subgroup of bacteriocins that are characterized by the presence of the unusual thioether amino acids lanthionine and 3-methyllanthionine generated through posttranslational modification. The biosynthesis of lantibiotics follows a defined pathway comprising modifications of the prepeptide, proteolytic activation, and export. The genes encoding the biosynthesis apparatus and the lantibiotic prepeptide are organized in clusters, which also include information for proteins involved in regulation and producer self-protection. The elongated cationic lantibiotics primarily act through the formation of pores and recent progress with nisin and epidermin has shown that specific docking molecules such as lipid II play an essential role in this mechanism. Mersacidin and actagardine inhibit cell wall biosynthesis by complexing the precursor lipid II, whereas the cinnamycin-like peptides bind to phosphoethanolamine thus inhibiting phospholipase A2. © 2000 John Wiley & Sons, Inc. Biopoly 55: 62–73, 2000

Posttranslationally modified bacteriocins--the lantibiotics.

Lantibiotics are a subgroup of bacteriocins that are characterized by the presence of the unusual thioether amino acids lanthionine and 3-methyllanthionine generated through posttranslational modification. The biosynthesis of lantibiotics follows a defined pathway comprising modifications of the prepeptide, proteolytic activation, and export. The genes encoding the biosynthesis apparatus and the lantibiotic prepeptide are organized in clusters, which also include information for proteins involved in regulation and producer self-protection. The elongated cationic lantibiotics primarily act through the formation of pores and recent progress with nisin and epidermin has shown that specific docking molecules such as lipid II play an essential role in this mechanism. Mersacidin and actagardine inhibit cell wall biosynthesis by complexing the precursor lipid II, whereas the cinnamycin-like peptides bind to phosphoethanolamine thus inhibiting phospholipase A2.

Lysinoalanine in food and in antimicrobial proteins.

Heat and alkali treatment of food proteins widely used in food processing results in the formation of crosslinked amino acids such as lysinoalanine, ornithinoalanine, lanthionine, and methyl-lanthionine and concurrent racemization of L-amino acid isomers to D-analogues. The mechanism of lysinoalanine formation is a two-step process: first, hydroxide ion-catalyzed elimination of cysteine and serine residues to a dehydroalanine intermediate; second, reaction of the double bond of dehydroalanine with the epsilon-NH2 group of lysine to form a lysinoalanine crosslink. The corresponding elimination-addition reaction of threonine produces methyl-dehydroalanine, which then reacts with the NH2 and SH groups to form methyl-lysinoalanine and methyl-lanthionine, respectively. The crosslinked amino acids lanthionine and methyl-lanthionine are formed by analogous nucleophilic addition reactions of the SH group of cysteine to dehydroalanine and methyl-dehydroalanine, respectively. Processing conditions that favor these transformations include high pH, temperature and exposure time. Factors which minimize lysinoalanine formation include the presence of SH-containing amino acids such as cysteine, N-acetyl-cysteine, and glutathione, dephosphorylation of O-phosphoryl esters, and acylation of epsilon-NH2 groups of lysine side chains. The presence of lysinoalanine residues along a protein chain decreases digestibility and nutritional quality in rodents but enhances nutritional quality in ruminants. Protein-bound and free lysinoalanines are reported to induce enlargement of nuclei of rat kidney cells. All of the mentioned dehydro and crosslinked amino acids also occur naturally in certain peptide and protein antibiotics. These include duramycin, cinnamycin, epidermin, subtilin and the widely used food preservative nisin. Mechanistic rationalizations are offered for the observed antimicrobial activities of these compounds in relation to their structures. The cited findings and new research to better define the chemistry and dietary and antimicrobial roles of lysinoalanine and related compounds should lead to better and safer foods.

The biosynthesis of the lantibiotics epidermin, gallidermin, Pep5 and epilancin K7.

Lantibiotics are antibiotic peptides that contain the rare thioether amino acids lanthionine and/or methyllanthionine. Epidermin, Pep5 and epilancin K7 are produced by Staphylococcus epidermidis whereas gallidermin (6L-epidermin) was isolated from the closely related species Staphylococcus gallinarum. The biosynthesis of all four lantibiotics proceeds from structural genes which code for prepeptides that are enzymatically modified to give the mature peptides. The genes involved in biosynthesis, processing, export etc. are found in gene clusters adjacent to the structural genes and code for transporters, immunity functions, regulatory proteins and the modification enzymes LanB, LanC and LanD, which catalyze the biosynthesis of the rare amino acids. LanB and LanC are responsible for the dehydration of the serine and threonine residues to give dehydroalanine and dehydrobutyrine and subsequent addition of cysteine SH-groups to the dehydro amino acids which results in the thioether rings. EpiD, the only LanD enzyme known so far, catalyzes the oxidative decarboxylation of the C-terminal cysteine of epidermin which gives the C-terminal S-aminovinylcysteine after addition of a dehydroalanine residue.

Engineering of a novel thioether bridge and role of modified residues in the lantibiotic Pep5

Pep5 is a 34-amino-acid antimicrobial peptide, produced by Staphylococcus epidermidis 5, that contains the thioether amino acids lanthionine and methyllanthionine, which form three intramolecular ring structures. In addition, two didehydrobutyrines are present in the central part of the lantibiotic and an oxobutyryl residue is located at the N terminus. All rare amino acids are introduced by posttranslational modifications of a ribosomally made precursor peptide. To elucidate the function of the modified residues for the antimicrobial action of Pep5, mutant peptides, in which single modified residues had been eliminated, were produced by site-directed mutagenesis. All of these peptides showed a reduced antimicrobial activity. In addition, those peptides from which the ring structures had been deleted became susceptible to proteolytic digest. This demonstrates that the ring structures serve as stabilizers of conformations essential for activity, e.g., amphiphilicity, as well as for protecting Pep5 against proteases of the producing strains. In addition, residues that could serve as precursors of new modified amino acids in lantibiotics were introduced into the Pep5 precursor peptide. This way, a novel methyllanthionine and a didehydroalanine were inserted into the flexible central part of Pep5, demonstrating that biosynthesis of modified amino acids is feasible by protein engineering and use of the lantibiotic modification system.

Isolation and characterization of genetically engineered gallidermin and epidermin analogs.

Gallidermin (Gdm) and epidermin (Epi) are highly homologous tetracyclic polypeptide antibiotics that are ribosomally synthesized by a Staphylococcus gallinarum strain and a Staphylococcus epidermidis strain, respectively. These antibiotics are secreted into media and are distinguished by the presence of the unusual amino acids lanthionine, 3-methyllanthionine, didehydrobutyrine, and S-(2-aminovinyl)-D-cysteine, which are formed by posttranslational modification. To study the substrate specificities of the modifying enzymes and to obtain variants that exhibit altered or new biological activities, we changed certain amino acids by performing site-specific mutagenesis with the Gdm and Epi structural genes (gdmA and epiA, respectively). S. epidermidis Tü3298/EMS6, an epiA mutant of the Epi-producing strain, was used as the expression host. This mutant synthesized Epi, Gdm, or analogs of these antibiotics when the appropriate genes were introduced on a plasmid. No Epi or Gdm analogs were isolated from the supernatant when (i) hydroxyamino acids involved in thioether amino acid formation were replaced by nonhydroxyamino acids (S3N and S19A); (ii) C residues involved in thioether bridging were deleted (delta C21, C22 and delta C22); or (iii) a ring amino acid was replaced by an amino acid having a completely different character (G10E and Y20G). A strong decrease in production was observed when S residues involved in thioether amino acid formation were replaced by T residues (S16T and S19T). A number of conservative changes at positions 6, 12, and 14 on the Gdm backbone were tolerated and led to analogs that had altered biological properties, such as enhanced antimicrobial activity (L6V) or a remarkable resistance to proteolytic degradation (A12L and Dhb14P). The T14S substitution led to simultaneous production of two Gdm species formed by incomplete posttranslational modification (dehydration) of the S-14 residue. The fully modified Dhb14Dha analog exhibited antimicrobial activity similar to that of Gdm, whereas the Dhb14S analog was less active. Both peptides were more sensitive to tryptic cleavage than Gdm was.