Papers Highlight Research on Antibiotic Biosynthesis

Andrew M. Gulick, PhD

Published October 23, 2019

story by bill bruton

Research by Andrew M. Gulick, PhD, associate professor of structural biology, that solved the structure of two proteins that produce antibiotic agents is featured in papers in back-to-back months of Nature Communications.

“Over the last three or four years, my lab and a couple of others have been able to identify structures of these large multi-domain assembly-line proteins to really start to understand the transit from one active site to the next.”
Associate professor of structural biology

Protein Structures of NRPS Systems

A paper published in July details the X-ray crystal structure of the enzyme that makes obafluorin, a β-lactone antibiotic agent made by a fluorescent strain of soil bacteria.

A paper published in August deals with the X-ray crystal structure of the enzyme that makes nocardicin, a monocyclic β-lactam antibiotic.

“They’re both produced by the same assembly line family of proteins: non-ribosomal peptide synthetases (NRPSs),” says Gulick, senior author of both papers.

Gulick has a long history of the study of NRPS systems. In two decades of research, he has published many of the important crystal structures of core pieces of these enzymes, along with structures for full-length systems. These structures have guided the understanding of how these enzymes work and how they can be modified to produce new antibiotics.

Single- and Multi-Domain Structures Tackled

Like penicillin, obafluorin has a four-membered ring sometimes referred to as an enchanted ring.

“With the obafluorin NRPS, we solved a large, multi-domain structure that gives insights into this assembly line system. With the nocardicin NRPS, we solved a single-domain structure of the protein which gives insight into the chemistry,” Gulick says.

Gulick collaborated with Timothy Wencewicz, PhD, associate professor of chemistry at Washington University in St. Louis, for research on obafluorin. Obafluorin was discovered in 1984, but it wasn’t until 2017 that the Wencewicz lab described the genetic blueprint of the enzyme that makes the molecule’s bio-active components.

That marked the first time researchers had been able to pin down a β-lactone forming enzyme from nature, and recreate it in a laboratory.

“The protein structure that we solved in collaboration with Dr. Wencewicz’s lab showed how the enzyme recognizes its building blocks, and how important features like the lactone ring are formed,” Gulick says. “The Wencewicz lab performed some elegant studies to make variants of this obafluorin molecule that might have better properties. We are hoping that these insights might allow the identification of other related lactones or molecules that could serve as antibiotics that we don’t know anything about yet.”

Protein Structure Has ‘Assembly-Line Process’

Gulick says this family of proteins is rather unique in how they function.

“Our structures show the assembly-line process at work. In most biosynthetic pathways, each protein does one chemical step and then releases the product. This product then finds another protein that does the next step,” Gulick says. “These NRPS proteins have all the catalytic or functional parts of the protein joined in one single, very large protein. Instead of the substrate or the molecule floating from site to site through solution, it’s actually bound to the protein.”

“Over the last three or four years, my lab and a couple of others have been able to identify structures of these large multi-domain assembly-line proteins to really start to understand the transit from one active site to the next,” Gulick says.

Because the antibiotic products are bound to the proteins, another enzymatic activity is necessary to release the final product. Gulick collaborated with Craig Townsend, PhD, Alsoph H. Corwin Professor of chemistry at Johns Hopkins University, to examine the domain that catalyzes this step in nocardicin biosynthesis. “While nearly all NRPSs end with a domain that releases the product, a thioesterase domain, the nocardicin NRPS has an unusual bifunctional domain that does an additional chemical step before release. We were able to use a very clever analog designed by Dr. Townsend and his colleagues to get a view of this process.”

Structural Studies Serve Dual Purpose

The goal of the research is to provide the foundation for studies to engineer NRPS enzymes to create a chemical library, populated with related β-lactone compounds that have been engineered for useful purposes. Additionally, it may be possible to find new NRPS systems that make entirely unknown molecules.

“We’re interested in how bacteria produce natural products. About one-half or two-thirds of the current FDA-approved drugs are either natural products or derived from natural products, which means it wasn’t something that some chemist dreamed up — it was produced by bacteria, yeast or fungus,” Gulick says. “My lab is interested in how the bacteria produce these natural products. If you knew that, you could produce new ones, or you could engineer them. A lot of these molecules are extremely complicated and hard for a chemist to make, so we’re trying to exploit the bacterial systems to do that.”

Dale Kreitler, PhD, and Ketankumar D. Patel, PhD, postdoctoral associates in the Department of Structural Biology working in Gulick’s lab, were the lead authors on the papers. Kreitler studied the structure of the obafluorin NRPS and Patel performed the structural studies of the nocardicin thioesterase.