19 research outputs found
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Non-Proteinogenic Amino Acids in Lacticin 481 Analogues Result in More Potent Inhibition of Peptidoglycan Transglycosylation
Lantibiotics are ribosomally synthesized and post-translationally modified peptide natural products that contain the thioether structures lanthionine and methyllanthionine and exert potent antimicrobial activity against Gram-positive bacteria. At present, detailed modes-of-action are only known for a small subset of family members. Lacticin 481, a tricyclic lantibiotic, contains a lipid II binding motif present in related compounds such as mersacidin and nukacin ISK-1. Here, we show that lacticin 481 inhibits PBP1b-catalyzed peptidoglycan formation. Furthermore, we show that changes in potency of analogues of lacticin 481 containing non-proteinogenic amino acids correlate positively with the potency of inhibition of the transglycosylase activity of PBP1b. Thus, lipid II is the likely target of lacticin 481, and use of non-proteinogenic amino acids resulted in stronger inhibition of the target. Additionally, we demonstrate that lacticin 481 does not form pores in the membranes of susceptible bacteria, a common mode-of-action of other lantibiotics.Chemistry and Chemical Biolog
In Vitro Mutasynthesis of Lantibiotic Analogues Containing Nonproteinogenic Amino Acids
In Vitro Mutasynthesis of Lantibiotic Analogues Containing Nonproteinogenic Amino Acid
NMR Structure of the SâLinked 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 6â16 and 26â35, and the
loop region encompasses residues 17â25. The 9-amino-acid loop
region contains a ÎČ-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
Development and Validation of a Multiplexed Protein Quantitation Assay for the Determination of Three Recombinant Proteins in Soybean Tissues by Liquid Chromatography with Tandem Mass Spectrometry
Currently,
traditional immunochemistry technologies such as enzyme-linked
immunosorbent assays (ELISA) are the predominant analytical tool used
to measure levels of recombinant proteins expressed in genetically
engineered (GE) plants. Recent advances in agricultural biotechnology
have created a need to develop methods capable of selectively detecting
and quantifying multiple proteins in complex matrices because of increasing
numbers of transgenic proteins being coexpressed or âstackedâ
to achieve tolerance to multiple herbicides or to provide multiple
modes of action for insect control. A multiplexing analytical method
utilizing liquid chromatography with tandem mass spectrometry (LC-MS/MS)
has been developed and validated to quantify three herbicide-tolerant
proteins in soybean tissues: aryloxyalkanoate dioxygenase (AAD-12),
5-enol-pyruvylshikimate-3-phosphate synthase (2mEPSPS), and phosphinothricin
acetyltransferase (PAT). Results from the validation showed high recovery
and precision over multiple analysts and laboratories. Results from
this method were comparable to those obtained with ELISA with respect
to protein quantitation, and the described method was demonstrated
to be suitable for multiplex quantitation of transgenic proteins in
GE crops
An Engineered Lantibiotic Synthetase That Does Not Require a Leader Peptide on Its Substrate
Ribosomally synthesized and post-translationally modified
peptides
are a rapidly expanding class of natural products. They are typically
biosynthesized by modification of a C-terminal segment of the precursor
peptide (the core peptide). The precursor peptide also contains an
N-terminal leader peptide that is required to guide the biosynthetic
enzymes. For bioengineering purposes, the leader peptide is beneficial
because it allows promiscuous activity of the biosynthetic enzymes
with respect to modification of the core peptide sequence. However,
the leader peptide also presents drawbacks as it needs to be present
on the core peptide and then removed in a later step. We show that
fusing the leader peptide for the lantibiotic lacticin 481 to its
biosynthetic enzyme LctM allows the protein to act on core peptides
without a leader peptide. We illustrate the use of this methodology
for preparation of improved lacticin 481 analogues containing non-proteinogenic
amino acids