In Vitro Mutasynthesis of Lantibiotic Analogues Containing Nonproteinogenic Amino Acids

In Vitro Mutasynthesis of Lantibiotic Analogues Containing Nonproteinogenic Amino Acid

NMR Structure of the S‑Linked Glycopeptide Sublancin 168

Sublancin 168 is a member of a small group of glycosylated antimicrobial peptides known as glycocins. The solution structure of sublancin 168, a 37-amino-acid peptide produced by <i>Bacillus subtilis</i> 168, has been solved by nuclear magnetic resonance (NMR) spectroscopy. Sublancin comprises two α-helices and a well-defined interhelical loop. The two helices span residues 6–16 and 26–35, and the loop region encompasses residues 17–25. The 9-amino-acid loop region contains a β-S-linked glucose moiety attached to Cys22. Hydrophobic interactions as well as hydrogen bonding are responsible for the well-structured loop region. The three-dimensional structure provides an explanation for the previously reported extraordinary high stability of sublancin 168

Haloduracin α Binds the Peptidoglycan Precursor Lipid II with 2:1 Stoichiometry

The two-peptide lantibiotic haloduracin is composed of two post-translationally modified polycyclic peptides that synergistically act on Gram-positive bacteria. We show here that Halα inhibits the transglycosylation reaction catalyzed by PBP1b by binding in a 2:1 stoichiometry to its substrate lipid II. Halβ and the mutant Halα-E22Q were not able to inhibit this step in peptidoglycan biosynthesis, but Halα with its leader peptide still attached was a potent inhibitor. Combined with previous findings, the data support a model in which a 1:2:2 lipid II:Halα:Halβ complex inhibits cell wall biosynthesis and mediates pore formation, resulting in loss of membrane potential and potassium efflux

The Glycosyltransferase Involved in Thurandacin Biosynthesis Catalyzes Both O- and S‑Glycosylation

The S-glycosyltransferase SunS is a recently discovered enzyme that selectively catalyzes the conjugation of carbohydrates to the cysteine thiol of proteins. This study reports the discovery of a second S-glycosyltransferase, ThuS, and shows that ThuS catalyzes both S-glycosylation of the thiol of cysteine and O-glycosylation of the hydroxyl group of serine in peptide substrates. ThuS-catalyzed S-glycosylation is more efficient than O-glycosylation, and the enzyme demonstrates high tolerance with respect to both nucleotide sugars and peptide substrates. The biosynthesis of the putative products of the <i>thuS</i> gene cluster was reconstituted <i>in vitro</i>, and the resulting S-glycosylated peptides thurandacin A and B exhibit highly selective antimicrobial activity toward <i>Bacillus thuringiensis.</i

Development, Validation, and Interlaboratory Evaluation of a Quantitative Multiplexing Method To Assess Levels of Ten Endogenous Allergens in Soybean Seed and Its Application to Field Trials Spanning Three Growing Seasons

As part of the regulatory approval process in Europe, comparison of endogenous soybean allergen levels between genetically engineered (GE) and non-GE plants has been requested. A quantitative multiplex analytical method using tandem mass spectrometry was developed and validated to measure 10 potential soybean allergens from soybean seed. The analytical method was implemented at six laboratories to demonstrate the robustness of the method and further applied to three soybean field studies across multiple growing seasons (including 21 non-GE soybean varieties) to assess the natural variation of allergen levels. The results show environmental factors contribute more than genetic factors to the large variation in allergen abundance (2- to 50-fold between environmental replicates) as well as a large contribution of Gly m 5 and Gly m 6 to the total allergen profile, calling into question the scientific rational for measurement of endogenous allergen levels between GE and non-GE varieties in the safety assessment

Characterization of Aryloxyalkanoate Dioxygenase-12, a Nonheme Fe(II)/α-Ketoglutarate-Dependent Dioxygenase, Expressed in Transgenic Soybean and <i>Pseudomonas fluorescens</i>

Aryloxyalkanoate dioxygenase-12 (AAD-12) was discovered from the soil bacterium <i>Delftia acidovorans</i> MC1 and is a nonheme Fe­(II)/α-ketoglutarate-dependent dioxygenase, which can impart herbicide tolerance to transgenic plants by catalyzing the degradation of certain phenoxyacetate, pyridyloxyacetate, and aryloxyphenoxypropionate herbicides. The development of commercial herbicide-tolerant crops, in particular AAD-12-containing soybean, has prompted the need for large quantities of the enzyme for safety testing. To accomplish this, the enzyme was produced in <i>Pseudomonas fluorescens</i> (<i>Pf</i>) and purified to near homogeneity. A small amount of AAD-12 was partially purified from transgenic soybean and through various analytical, biochemical, and <i>in vitro</i> activity analyses demonstrated to be equivalent to the <i>Pf</i>-generated enzyme. Furthermore, results from <i>in vitro</i> kinetic analyses using a variety of plant endogenous compounds revealed activity with <i>trans</i>-cinnamate and indole-3-acetic acid (IAA). The catalytic efficiencies (<i>k</i>_cat/<i>K</i>_m) of AAD-12 using <i>trans</i>-cinnamate (51.5 M^–1 s^–1) and IAA (8.2 M^–1 s^–1) as substrates were very poor when compared to the efficiencies of plant endogenous enzymes. The results suggest that the presence of AAD-12 in transgenic soybean would not likely have an impact on major plant metabolic pathways