Watching A Helper Protein Do Its Job

Acyl carrier protein (ACP) plays an essential role inside bacterial multicomponent enzyme systems. Like a little delivery truck, it transports substrates, intermediates, and products among catalytic domains as the enzyme systems biosynthesize natural products. And it uses a phosphopantetheine (Ppant) group as a working “arm” to actually carry out these tasks.

Researchers have now devised a technique that monitors Ppant motions occurring in nanoseconds or picoseconds—orders of magnitude faster than could be detected previously. The study might lead to a better molecular understanding of the workings of ACP-containing enzyme systems such as polyketide synthases, nonribosomal peptide synthases, and fatty acid synthases. That understanding could also make it easier to engineer multienzyme systems to produce customized natural-product-like compounds for drug discovery.

While ACP makes its rounds, the Ppant arm keeps changing shape to interact with individual enzyme units and help protect reactive intermediates from side reactions. The flexibility of the arm and the fleeting nature of its interactions make these motions difficult to detect. NMR and crystallography have been used to study Ppant arm motions, but NMR can’t analyze conformational changes that occur in less than a microsecond, and crystallography gives only static snapshots of arm conformations.

Undergraduate Matthew N. R. Johnson and chemistry professors Casey H. Londergan and Louise K. Charkoudian at Haverford College, in Pennsylvania, now report that installing a thiocyanate group on the end of the Ppant arm makes it possible to use infrared spectroscopy to analyze high-speed arm motions (J. Am. Chem. Soc. 2014, DOI: 10.1021/ja505442h). The technique follows the dynamics of the Ppant arm by monitoring vibrations of the thiocyanate’s nitrile bond.

“Sample preparation is quite easy, and not a lot of protein is required,” Charkoudian says. She and her coworkers used the technique to confirm a key part of the mechanism of 6-deoxyerythronolide B synthase for the first time experimentally.

“This is an innovative approach that transcends the traditional techniques such as protein NMR and crystallography for detecting the highly mobile Ppant arm in action,” comments polyketide biosynthesis expert Shiou-Chuan (Sheryl) Tsai of the University of California, Irvine.

“This cleverly designed experiment takes advantage of the sensitivity of the nitrile stretching vibration to its immediate environment and uses it to report on protein conformational changes that are important for function,” says mechanistic spectroscopist Feng Gai of the University of Pennsylvania. “It nicely demonstrates the power of combining a site-specific vibrational probe and infrared spectroscopy to yield mechanistic insights into enzymatic reactions.”