Princeton University has discovered a new pathway for antimicrobial peptides

Researchers in Princeton's Department of Chemistry have discovered a new multi-step pathway through which bacteria in the mammalian gut produce antimicrobial peptides
.

The newly discovered biosynthetic pathway converts a bioinert peptide into a structurally complex antibiotic, which they call an intestinal peptide
.
Intestinal peptides are a class of natural peptide products synthesized by ribosomes, referred to as RiPPs
.

The core structure of these products is synthesized by ribosomes, which are limited to 20 typical amino acids
.
The Mo lab discovered and characterized a new metalloenzyme capable of converting arginine, a typical amino acid, to N-methylmethnithine, an atypical amino acid
in intestinal peptides.

This is the first report
that RiPP natural products contain this unusual amino acid.
The discovery was made by the lab of Professor Mohammad Seyedsayamdost
.

Kenzie Clark, the paper's lead author and a former graduate student in Mo's lab, explains the core findings
.

"The way this pathway works is that ribosomes produce precursor peptides, which are then acted
by metalloenzymes encoded in the same gene cluster," Clark said.
"This series of metalloenzymes — in this case, three — converts arginine to N-methanornithine to produce intestinal peptides
in a step-by-step manner.

"Interestingly, the peptide itself does not show any biological activity
.
But once you add these modifications, it turns into this active biomolecule that can effectively inhibit the growth
of the production strain.
”

Brett Covington, a postdoctoral researcher at the lab and co-author of the paper, commented on the findings
.

"This is a trend we're seeing from many of the RiPP products we've found: they have a very narrow spectrum of activity and tend to inhibit the growth
of organisms that make compounds," he said.
"This is the case
with intestinal peptides.
It simply inhibits enterococci, which produces intestinal peptides
.
Why these Enterococci make an antibiotic peptide to inhibit their own growth is something
we're looking at.
They may be associated with
the development of more stubborn bacterial populations.
”

Work through gene clusters

This research fits into the mission of Mo's lab, which seeks to discover new bacterial natural products and understand how these products are biosynthesized
.

Back in 2018, in a study in the Journal of the American Chemical Society (JACS), Mo's lab used bioinformatics to discover 600 RiPP gene clusters in streptococci that utilize the free radical s-adenosylmethionine (rSAM) enzyme, one of
the largest families of enzymes known to nature.
Although it is a large group of more than 500,000 members that exists in all three kingdoms of life, most of these enzymes are not well understood
.

The lab grouped their newly discovered 600 RiPP gene clusters into 16 subfamilies
based on similar precursor peptide sequences.
They began studying these families in the lab, discovering new reactions and interesting chemistry along the way
.

"It's a new way to discover natural products, where you start with biosynthetic gene clusters and then do an in-depth analysis of the reaction of each enzyme to understand all the different transformations
that are happening," Covington said.
"We then looked for a mature product in the bacterial host, which contains all these different enzymatic transformations
.
This is a unique approach
.
”

So far, they have only studied clusters encoding a single metalloenzyme
.
The latest study targets a multi-step reaction pathway that contains more complex chemical reactions, not just one interesting reaction, but three reactions catalyzed by three different kinds of metalloenzymes
.
Two rely on iron and one on manganese
.

"One of the metalloenzymes produces a very active intermediate capable of very challenging chemical reactions, such as the formation of carbon-carbon bonds
in the center of inactive carbon," Clark said.
"Many of the structures they produce are very challenging for synthetic chemists
.

"But what's also interesting is the third enzyme
in that pathway.
" It is not annotated as a known family
of enzymes.
Finally, 4 iron 4 sulfur groups and the cofactor SAMN-methylornithine were used, which is a novel and interesting modification reaction
.
”

Covington added: "It's one of the things I enjoy the most when I work at Mo Labs, the excitement
you feel when you finally discover one of these products.
All the work is done on the bench to understand the reaction in the test tube, and then it's really comforting
when you see that it matches the reaction inside the actual bacteria.
”