Acyl carrier proteins (ACPs) are more important than they sound. Their name suggests they are simply biochemical beasts of burden. They indeed do carry substrates around and deliver them to different enzymes in bacteria and eukaryotes alike. But by fulfilling that task they play a crucial role in the biosyntheses of biomolecules essential for all organisms—such as fatty acids (biological fuel sources), polyketides (metabolite natural products on which a number of drugs are based), and lipoic acid and biotin (enzyme cofactors).
In bacteria, moreover, ACPs help coordinate the biosynthesis of lipid A, which is an indispensable component of the cell envelope and is toxic to hosts during infection. Lipid A is involved in sepsis, a severe infection that kills many hospital patients. A better understanding of its biosynthetic pathway could have drug discovery implications.
The way ACPs deliver substrates to enzymes is understood in its broad outlines. But the details remain cloudy, and scientists would like to know more of the particulars about how these processes work. Two new studies on the role of bacterial ACP—in biosyntheses of fatty acids and of lipid A, respectively—take major steps in that direction.
In bacteria, separate enzymes swimming around in the cytosol work together to make biomolecules such as fatty acids and lipid A. In each step of the biosynthetic sequence, ACP picks up the necessary substrates from, and delivers them to, the appropriate enzymes.
Researchers would like to better understand how ACP recognizes and binds to the correct enzymes, delivers its cargo to their active sites, picks up products, and detaches to make subsequent deliveries. But ACP’s interactions with each enzyme are only fleeting and thus hard to observe and analyze. ACP’s structural floppiness and flexibility have added to the difficulty.
Now, two groups have advanced the state of the art of ACP mechanistic analysis in different ways. One group consists of Shiou-Chuan (Sheryl) Tsai of the University of California, Irvine; Michael D. Burkart of UC San Diego; and coworkers, including UCSD’s J. Andrew McCammon and Stanley J. Opella. They used a cross-linker to trap the normally transient complex between bacterial ACP and the fatty acid dehydratase FabA (Nature 2013, DOI: 10.1038/nature12810). This enabled them to structurally analyze the interaction and propose details of some key steps in bacterial fatty acid synthesis.
The other group consists of Charles W. Pemble IV and coworkers at Duke University Medical Center, including grad student Ali Masoudi from the lab of the late Christian R. H. Raetz. They captured several crystal structures of ACP bound to the acyltransferase LpxD, an unusually stable ACP-enzyme complex (Nature 2013, DOI: 10.1038/nature12679). This allowed them to delineate new mechanistic aspects of a lipid A biosynthetic step at an unprecedented level of detail.
The complementary studies “give us a much better idea of how ACP reactions work,” comments enzymologist John Crosby of the University of Bristol, in England. The new insights aid understanding of ACP-based biosynthetic pathways, which play a key role in bacterial metabolism, and could lead to novel bacterial targets for antibiotic drug discovery.
Tsai, Burkart, and coworkers focused on the role of ACP and FabA in bacterial fatty acid synthesis. They designed a cross-linker that stabilizes the normally fleeting ACP-FabA complex. They then used X-ray crystallography, nuclear magnetic resonance spectrometry, and theoretical simulations to visualize the resulting protein-protein interactions, which had never been observed in such high detail before.
The complex crystallized as a dimer. In that dimer, the researchers were surprised to find, ACP is bound to FabA in two completely different ways. The two interactions, they believe, represent mechanistic snapshots of two parts of the reaction mechanism. The study suggests, for example, that in the later stages of substrate delivery, ACP repositions one of its helices to release the substrate and move it into the FabA active site. “That step was always speculated on, but nobody had really seen it in action,” says protein NMR specialist Hans Vogel of the University of Calgary.
“The work may be important in discovering new antibiotics that selectively target fatty acid biosynthesis by blocking these protein-protein interactions,” Burkart notes. He says it also might aid the design of pathways for the bacterial or algal production of improved biofuels.
Pemble and coworkers focused on the role of ACP in lipid A production. They determined three X-ray crystal structures of modified forms of ACP bound to the acyltransferase LpxD. The work revealed how ACP recognizes LpxD and maneuvers during the reaction. And it showed how ACP’s phosphopantetheine arm initiates a major structural change that provokes product release and initiates collapse of ACP-LpxD interactions.
The study found that “the carrier protein is effectively controlling significant stages of the catalytic cycle, which is quite new,” Crosby says.
Because ACP binding to LpxD is an unusually strong interaction, “it will be so exciting to see if structural reorganizations in the ACP-enzyme complex as observed in the study are also relevant for the majority of enzymes that use ACP,” comments structural biologist Timm Maier of the University of Basel.
“Targeting enzymes within the lipid A pathway represents an important therapeutic approach for the development of novel antibiotics,” Pemble says.
Next, researchers hope to visualize an early-stage ACP-enzyme complex, prior to ACP expelling its substrate cargo, and to study related ACPs, such as those integrated into multienzyme complexes in higher eukaryotes. Such studies could lead to the design of new “unnatural products,” Burkart notes. Pemble suggests they also could “drive the development of innovative drugs that improve human health.”