Nav: Home

Scientists capture the elusive structure of essential digestive enzyme

May 25, 2016

Using a powerful combination of techniques from biophysics to mathematics, researchers have revealed new insights into the mechanism of a liver enzyme that is critical for human health. The enzyme, phenylalanine hydroxylase, turns the essential amino acid phenylalanine -- found in eggs, beef and many other foods and as an additive in diet soda -- into tyrosine, a precursor for multiple important neurotransmitters.

"We need phenylalanine hydroxylase to control levels of phenylalanine in the blood because too much is toxic to the brain," said Steve Meisburger, lead author on the study and a post-doctoral researcher in the Ando lab. Genetic mutations in phenylalanine hydroxylase can lead to disorders such as phenylketonuria, an inherited condition that can cause intellectual and behavioral disabilities unless detected at birth and managed through dietary restrictions.

Published in the Journal of the American Chemical Society, the article presented detailed structural data on the enzyme's active state -- the shape it adopts when performing its chemical duties -- that has eluded scientists for years.

"It's a floppy enzyme which means it's dynamic," said Nozomi Ando, an assistant professor of chemistry at Princeton and corresponding author on the paper. "That also means it doesn't like to crystallize," she said. This is problematic for the classic method used to study enzymatic structure, known as x-ray crystallography, which requires solid crystal samples. Efforts to crystallize phenylalanine hydroxylase have just recently met success, but still only captured the enzyme in its inactive state.

The researchers in the Ando lab were able to bypass the tricky task of growing crystals of the active enzyme by using their expertise in a special technique akin to crystallography, called small angle x-ray scattering (SAXS), which allows scientists to study enzymes in a solution. And because the enzyme is susceptible to aggregation or clumping up in solution, the researchers coupled their scattering method with a purification technique called size exclusion chromatography (SEC), in which different species in a sample flow through a column at different speeds based on their size.

"Pairing SEC with SAXS is an emergent technique. Our contribution is that we saw a clever way to use it," Ando said. The experiment is highly specialized and relies on powerful x-rays emitted by particles speeding around the circular track at a synchrotron facility. The research team traveled from Princeton to the Cornell High Energy Synchrotron Source in Ithaca, New York, for multiple intensive data-collection sessions. "Any time on the machine that is available, we use it. Not a single photon gets wasted," Ando said.

As the enzyme solution passes through the purification technique, flowing across the path of the x-ray beam, researchers record snapshots of the x-ray scattering patterns. The resulting dataset is quite complex as the sample also contains phenylalanine, the compound that "turns on" phenylalanine hydroxylase so that researchers can catch the dynamic enzyme in action.

"Current approaches for analyzing this type of dataset are very crude," Meisburger said. Essentially, these methods assume that each signal -- known as an elution peak -- represents a single species, when each peak is actually a mixture of species. In this work, the team used an advanced linear algebra method known as evolving factor analysis that allowed them to separate the scattering components. "We can use these linear algebra methods to 'un-mix' species that are overlapping," Meisburger said, "That's the piece that I think is really exciting."

By applying their unique approach, the researchers were able to provide evidence for a model of the active structure of phenylalanine hydroxylase that builds upon recent work by their collaborators in Paul Fitzpatrick's group at UT Health Science Center at San Antonio. In this model, two phenylalanine molecules dock to a pair of sites on the enzyme, bringing a pair of arms together and freeing up the active sites for doing chemistry once more phenylalanine molecules come along.

"I'm very proud that this is our first paper [published since Ando joined the faculty at Princeton]. We wanted it to be very quantitative and heavy on the biochemistry plus heavy on the physical chemistry. I'm really pleased with the way it turned out," Ando said.
-end-
Read the full paper here:

Meisburger, S. P.; Taylor, A. B.; Khan, C. A.; Zhang, S.; Fitzpatrick, P. F.; Ando, N. "Domain movements upon activation of phenylalanine hydroxylase characterized by crystallography and chromatography-coupled small-angle X-ray scattering." J. Am. Chem. Soc. 2016 Article ASAPS

This work was supported by National Health Institutes grants GM100008 and GM098140 and Welch Foundation grant AQ-1245.

Princeton University

Related Enzyme Articles:

Enzyme catalyzed decomposition of 4-hydroxycyclophosphamide
Oxazaphosphorine cytostatics (Cyclophosphamide, Ifosfamide) are often used and very effective anticancer agents; but so far little is known about the molecular basis for the antitumor effect.
The carpenter enzyme gives DNA the snip
Enzyme follows a two-step verification system before cutting and repairing DNA damage.
Cellular senescence prevented by the SETD8 enzyme
An enzyme that blocks cellular senescence and its mechanisms has been discovered by a Japanese research team.
Enzyme key to learning in fruit flies
University of California, Riverside-led research finds enzyme that is key to learning in fruit flies.
Old enzyme, new role
A team of researchers at the University of Delaware has discovered a new function for an enzyme that has long been known to have a central role in bacterial metabolism.
Enzyme research provides a new picture of depression
Depression is the predominant mental disease and constitutes the most common cause of morbidity in developed countries.
Mysteries of enzyme mechanism revealed
International team led by University of Leicester unveil a hidden step in enzyme mechanism.
Single enzyme controls 2 plant hormones
Scientists at Washington University in St. Louis have isolated the first enzyme shown to be capable of controlling the levels of two distinct plant hormones, involved both in normal growth and in responses to infections.
New enzyme-mapping advance could help drug development
Scientists at MIT and the University of São Paulo in Brazil have identified the structure of an enzyme that could be a good target for drugs combatting three diseases common in the developing world.
Severity of enzyme deficiency central to favism
The congenital disease favism causes sickness and even jaundice in patients after they consume beans.

Related Enzyme Reading:

Best Science Podcasts 2019

We have hand picked the best science podcasts for 2019. Sit back and enjoy new science podcasts updated daily from your favorite science news services and scientists.
Now Playing: TED Radio Hour

Setbacks
Failure can feel lonely and final. But can we learn from failure, even reframe it, to feel more like a temporary setback? This hour, TED speakers on changing a crushing defeat into a stepping stone. Guests include entrepreneur Leticia Gasca, psychology professor Alison Ledgerwood, astronomer Phil Plait, former professional athlete Charly Haversat, and UPS training manager Jon Bowers.
Now Playing: Science for the People

#524 The Human Network
What does a network of humans look like and how does it work? How does information spread? How do decisions and opinions spread? What gets distorted as it moves through the network and why? This week we dig into the ins and outs of human networks with Matthew Jackson, Professor of Economics at Stanford University and author of the book "The Human Network: How Your Social Position Determines Your Power, Beliefs, and Behaviours".