Piperazic acid (Piz) is a nonproteinogenic amino acid
possessing
a rare nitrogen–nitrogen bond. However, little is known about
how Piz is incorporated into nonribosomal peptides, including whether
adenylation domains specific to Piz exist. In this study, we show
that free piperazic acid is directly adenylated and then incorporated
into the incarnatapeptin nonribosomal peptides through isotopic incorporation
studies. We also use in vitro reconstitution to demonstrate
adenylation of free piperazic acid with a three-domain nonribosomal
peptide synthetase from the incarnatapeptin gene cluster. We furthermore
use bioinformatics and site-directed mutagenesis to outline consensus
sequences for the adenylation of piperazic acid, which can now be
used for the prediction of gene clusters linked to piperazic-acid-containing
peptides. Finally, we discover a fusion protein of a piperazate synthase
and an adenylation domain, highlighting the close biosynthetic relationship
of piperazic acid formation and its adenylation. Altogether, our work
demonstrates the evolution of biosynthetic systems for the activation
of free piperazic acid through adenylation, a pathway we suggest is
likely to be employed in the majority of pathways to piperazic-acid-containing
peptides.
The biosynthesis, breakdown, and modification of peptidoglycan (PG) play vital roles in both bacterial viability and in the response of human physiology to bacterial infection. Studies on PG biochemistry are hampered by the fact that PG is an inhomogeneous insoluble macromolecule. Chemical synthesis is therefore an important means to obtain PG fragments that may serve as enzyme substrates and elicitors of the human immune response. This review outlines the recent advances in the synthesis and biochemical studies of PG fragments, PG biosynthetic intermediates (such as Park's nucleotides and PG lipids), and PG breakdown products (such as muramyl dipeptides and anhydro‐muramic acid‐containing fragments). A rich variety of synthetic approaches has been applied to preparing such compounds since carbohydrate, peptide, and phospholipid chemical methodologies must all be applied.
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