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Biological Chemistry

X-ray ‘Snapshots’ Reveal Details Of How Megaenzymes Synthesize Natural Products

Structural Biochemistry: Rare nonribosomal peptide synthetase crystal structures expose biosynthetic secrets

by Stu Borman
January 15, 2016

Credit: Nature
A peptidyl carrier protein (PCP) makes a 75° swing and moves 61 Å as it transitions between sites that add thiol groups and then formyl groups to an intermediate in an antibiotic-producing NRPS.
Protein ribbon model shows how a peptidyl carrier protein (PCP) domain makes a 75o swing and moves 61 Å as it transitions between thiol and formyl states in an antibiotic-producing NRPS.
Credit: Nature
A peptidyl carrier protein (PCP) makes a 75° swing and moves 61 Å as it transitions between sites that add thiol groups and then formyl groups to an intermediate in an antibiotic-producing NRPS.

Researchers have unveiled long-sought snapshots of the complex inner workings of nonribosomal peptide synthetases (NRPSs), megaenzymes that bacteria and fungi use to produce peptide-based natural products—including approved drugs such as the antibiotic vancomycin and the immunosuppressant cyclosporine. Companies take advantage of NRPSs by growing bacteria and fungi to pump out drugs such as these on a commercial scale.

NRPSs are assembly-line systems with multiple enzyme domains organized into modules that carry out sequential steps in complex biosynthetic processes. Scientists are trying to understand in detail how NRPSs work to be better able to rationally engineer the megaenzymes to produce customized peptide-based natural products as possible therapeutics. But three-dimensional structures that reveal mechanistic details of how NRPSs work are difficult to obtain and remain fairly scarce.

Two new X-ray crystallography studies now add color to researchers’ picture of the way NRPSs work. In one, T. Martin Schmeing and coworkers at McGill University obtained crystal structures of an antibiotic-producing NRPS module that includes a specialized unit that adds formyl groups to substrates (Nature 2016, DOI: 10.1038/nature16503). The structures show unusually large movements that module components make as they do their jobs. The most dramatic is a 75° rotation and 61-Å motion of a peptidyl carrier protein as it moves between catalytic sites that thiolate and then formylate a biosynthetic intermediate.

Andrew M. Gulick of Hauptman-Woodward Medical Research Institute and the University at Buffalo and coworkers determined two crystal structures of bacterial NRPS components that also reveal the way key catalytic steps occur (Nature 2016, DOI: 10.1038/nature16163). One crystal structure demonstrates a major conformational change in a domain that adds adenosine monophosphate to an intermediate and guides transfer of the intermediate between catalytic sites. Another crystal structure shows how condensation and adenylation sites adopt active catalytic conformations simultaneously, boosting the efficiency of the biosynthetic process. “By being able to perform two steps at once, the whole process is faster,” Gulick says.

“The papers aid understanding of the conformational dynamics of these NRPS machines,” comments Mohamed A. Marahiel of Philipps University Marburg, a specialist in NRPS reaction mechanisms. “Such dynamics were suggested before in earlier studies and therefore are not surprising. But it’s nice to see them confirmed experimentally, and both studies could have a significant influence” on the ability to rationally engineer NRPSs.

“There is very little structural information out there on NRPSs, and these studies make important contributions to that repertoire,” adds natural product expert Michael D. Burkart of the University of California, San Diego. To be able to engineer NRPSs to make novel metabolites and drug candidates, scientists will continue to need more studies like these to explain how the megaenzymes work, Burkart says.

Martin Schmeing discusses the rationale for his group’s new structural study on an antibiotic-producing NRPS and shows simulations of the megaenzyme’s inner workings.
Credit: McGill University Services


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