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.
Acyl-CoA: diacylglycerol acyltransferase (DGAT) catalyzes the final committed step in triacylglycerol (TAG) biosynthesis in eukaryotes. In microalgae, the copy number of DGAT genes is extraordinarily expanded and yet the functions of many DGATs remain largely unknown. This study revealed microalgal DGAT can function as lysophosphatidic acyltransferase (LPAAT) both in vitro and in vivo while losing its original function as DGAT. Among the five DGAT encoding genes identified and cloned from the green microalga Haematococcus pluvialis, four encodes HpDGATs that showed TAG synthase activities in yeast for functional complementation analysis, except for one of the type II DGAT encoding genes, i.e., HpDGTT2 gene. The hydrophobic recombinant HpDGTT2 was purified in a soluble form and was revealed to function as a LPAAT via enzymatic assay. Introducing this gene into the green microalga Chlamydomonas reinhardtii led to retarded cellular growth, enlarged cell size and enhanced TAG accumulation, identical to the phenotypes of the transgenic strains with overexpressed CrLPAAT. This study provides a framework for dissecting uncharacterized DGATs, and could pave the way to decrypt the structure-function relationship of this large group of enzymes critical to lipid biosynthesis.
Azaserine (1) is a natural product and nonproteinogenic
amino acid containing a diazo group. Here we report the biosynthetic
gene cluster for 1 from Glycomyces harbinensis. We then use isotopic feeding, gene deletion, and biochemical experiments
to support a pathway whereby hydrazinoacetic acid (2)
and a peptidyl carrier protein-loaded serine (3) are
intermediates on route to the final natural product 1.
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