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March 13, 2006
Volume 84, Number 11
p. 9


Nisin Engineered In Test Tube

Biosynthetic route may open up access to analogs of potent antibiotic

Amanda Yarnell

Photo by L. Brian Stauffer/UIUC

Cyclase Sleuths Nair, Li, van der Donk, and Yu teamed up to crack the biosynthesis of nisin and the structure of the key cyclase enzyme involved (shown on the computer screen).

Formerly inaccessible analogs of the potent antibiotic nisin-a complex peptide natural product used for more than 40 years to preserve food-may soon become available. Scientists at the University of Illinois, Urbana-Champaign (UIUC), have figured out how to harness the biosynthetic machinery that makes nisin so they can make it in vitro, potentially paving the way to versions that might be used to treat infections in humans (Science 2006, 311, 1464).

The growing problem of bacterial resistance has inspired many attempts to exploit bacterial biosynthetic machinery to create new analogs of antibiotics. Insight into the biosynthesis of nisin is of particular interest because "even though it's been widely used as a food preservative for many decades, significant bacterial resistance to nisin has not developed," notes UIUC chemist Wilfred A. van der Donk. He says bacteria have not been able to devise a way to resist nisin because the antibiotic packs an unusually deadly one-two punch, both disrupting bacterial cell wall biosynthesis and punching holes in bacterial cell membranes.

But despite nisin's nanomolar potency against a range of gram-positive bacteria, it isn't used to treat infections in humans because it isn't stable at the body's neutral pH and it is readily inactivated by physiological free thiols, notes John C. Vederas of the University of Alberta, Edmonton. He adds that "new active nisin analogs that are stable at neutral pH and are resistant to thiols would have great potential" in treating infections caused by drug-resistant bacteria. The UIUC team's work provides a basis for the design of such analogs, Vederas says.

Nisin is made naturally by Lactococcus lactis, a strain of bacteria that grows on food, particularly dairy products. The complex natural product contains 34 amino acids, including a laundry list of unusual ones, and five thioether rings of various sizes. "Synthetic chemists in the past needed 67 steps to make it, while nature uses just three enzymes," van der Donk says.

Courtesy of Wilfred van der Donk/UIUC

Loopy Nisin contains five different thioether (red) rings of varying sizes.

L. lactis uses one enzyme to dehydrate specific side chains on a linear precursor peptide. Bo Li and van der Donk isolated this dehydrated precursor peptide from an engineered strain of L. lactis. They then showed that a single cyclase enzyme called NisC can make all of nisin's rings in a test tube. This enzyme catalyzes five distinct cyclization reactions to generate nisin's five thioether rings, "a remarkable feat of both regio- and stereoselective control," van der Donk notes. A final trim by a protease gives the mature antibiotic.

The structure of NisC, solved by UIUC's John Paul J. Yu, Joseph S. Brunzelle, and Satish C. Nair, "illuminates fascinating aspects of the cyclization mechanism," writes David W. Christianson of the University of Pennsylvania in an accompanying commentary.

Nair says the structure strongly suggests that a zinc ion in the enzyme's active site activates each of five specific cysteines in the dehydrated precursor peptide in turn. The team is now working to figure out how the enzyme manages to make each ring in the right place and the right size. "We are also trying to feed novel substrates to NisC in order to make new nisin analogs," van der Donk says.

Courtesy of Satish Nair/UIUC

Ringmaster NisC catalyzes the formation of five distinct rings of varying sizes.

Chemical & Engineering News
ISSN 0009-2347
Copyright © 2010 American Chemical Society