Scientists at Ohio State University used mass spectrometry and a series of experiments to discover how cells make the amino acid, a process that until now had been unknown.
They confirmed that pyrrolysine is made from enzymatic reactions with two lysine molecules – a surprising finding, given that some portions of its structure suggested to researchers that it might have more complex origins.
The research is published in the March 31 issue of the journal Nature.
Pyrrolysine is rare and so far is known to exist in about a dozen organisms. But its discovery in 2002 as a genetically encoded amino acid in methane-producing microbes raised new questions about the evolution of the genetic code. Pyrrolysine is among 22 amino acids that are used to create proteins from the information stored in genes. Proteins are essential to all life and perform most of the work inside cells.
This information about how it is produced – its biosynthetic pathway – offers a more complete understanding of how amino acids are made. And because of its rarity, this molecule is emerging as a handy tool for manipulating proteins in biomedical research. With its production mechanism identified, scientists can use that information to devise ways to mass-produce similar or identical synthetic molecules for a variety of research purposes.
The Ohio State scientists had a genuine “ah-ha” moment over the course of the study. As part of their experimentation, they combined lysine with one other amino acid and some enzymes and expected this to produce what is called an intermediate – essentially, a piece of an amino acid that is generated in the biosynthesis process.
They had labeled the lysine so it would appear heavier than normal when observed using mass spectrometry. But one signal produced by the instrumentation had a much different mass than could be attributed to the intermediate.
“We weren’t seeing this weird molecule made from two different amino acids that we were expecting. We were seeing the regular pyrrolysine molecule and all of it was coming from lysine. Every bit of it,” said Joseph Krzycki, professor of microbiology at Ohio State and senior author of the study. “That was the only way we saw pyrrolysine, and all of it was labeled with lysine. That’s the basic observation here. And it’s a real surprise.”
The finding that lysine was the only precursor was a surprise because the production process ended up being so simple – even though arriving at it was not a simple task, partly because some of the chemical reactions had never been observed before.
“What amazes me about the entire chemical pathway is that you need only three enzymes and two molecules of the same thing that together make one complete molecule that looks completely different from what you started with,” said Marsha Gaston, first author of the paper and a doctoral student in microbiology. “You have one portion that looks exactly like the precursor, but then you have another portion that enzymes are able to re-arrange in a way that is completely unique and never seen before.”
Mass spectrometry, an analytic technique that enables precision in determining the mass of particles, ended up being critical to the discoveries, Krzycki noted. Liwen Zhang and Kari Green-Church of Ohio State’s Campus Chemical Instrument Center/Mass Spectrometry and Proteomics Facility are additional co-authors of the study.
Krzycki led one of the two teams of Ohio State researchers that discovered pyrrolysine in 2002. The teams have since synthesized the amino acid and shown how bacteria incorporate it into proteins.
“That left some big questions unanswered: How do you make pyrrolysine? Where does it come from? What metabolic pathways does it come off of? Because it’s got to be generated within the cell that uses it,” Krzycki said.
The chemical shape of pyrrolysine offered some clues. Its carbon skeleton resembles that of lysine. But it also has an unusual ring on one end, and a methyl group attached to it, which for researchers raised questions about its origin.
The researchers also knew from their previous work that three genes are required to generate the instructions for the assembly of proteins that contain pyrrolysine – pylB, pylC and pylD. So the enzymes produced by those three genes had to have a role in creation of the amino acid. Finally, previous attempts by other researchers to define its biosynthesis suggested that another amino acid, D-ornithine, was involved in pyrrolysine’s production.
So Krzycki and his colleagues set out to test that theory. Conducting all of their experiments in a strain of E. coli bacteria, commonly used to test biological functions, they combined lysine and D-ornithine molecules.
They found that this didn’t make pyrrolysine, but rather a molecule like pyrrolysine that was missing a key part; however, this molecule turned out not to be converted to pyrrolysine. This molecule also was formed without the involvement of pylB – a gene that could not be left out of the process that actually makes pyrrolysine.
With the mass spectrometry instead identifying lysine as the only precursor to pyrrolysine, the researchers then used genetics, mass spectrometry of intermediates and deduction to determine the order of enzymatic reactions that converted two lysine molecules into the pyrrolysine amino acid.
They determined that the sequence of events matched the alphabetical order of the three involved enzymes: PylB uses lysine to make a D-ornithine-like intermediate, PylC joins the two lysine molecules together, and that feeds a reaction involving PylD that results in the formation of pyrrolysine. The reactions showed how the ring on pyrrolysine’s end, its major identifying characteristic, is formed.
“If you splay out the pyrrolysine molecule, you can recognize that in fact it looks a lot like lysine, except that to get to this ring, you have to make the second molecule one carbon unit shorter,” Krzycki said. “The lysine goes through a type of enzymatic reaction called a mutase reaction, where the carbon skeleton is rearranged to make this shorter molecule, which is like D-ornithine, but with one extra carbon now hanging off the chain in a new place. That’s what one of our pyrrolysine biosynethetic enzymes, PylB, is doing.”
Krzycki noted that this finding will add fuel to discussions of how the genetic code evolved. For example, the co-evolutionary theory suggests that amino acids arising from a common precursor have similar codon assignments. Codons are three-letter “words” identifying the bases that DNA uses to specify particular amino acids as building blocks of proteins. Normally, codons signal the start or end of a protein, or a particular amino acid used to construct it.
“For the scientists who are devoted to exploring how the genetic code evolved, our data provides new insights that can feed the various theories for how the code evolved; the co-evolutionary theory is just one such example,” Krzycki said.
The finding that pyrrolysine derives entirely from lysine means that pyrrolysine is part of the aspartic acid family in bacteria and Archaea, a group of single-cell microorganisms that are similar to bacteria in size and shape, but with a different evolutionary history. The microbes known to contain pyrrolysine are in the Archaea domain, and are able to convert a common class of compounds – the methylamines – into methane gas.
This work was supported by grants from the National Institutes of Health and the U.S. Department of Energy.Contact: Joseph Krzycki, (614) 292-1578; email@example.com
Emily Caldwell | Newswise Science News
The balancing act: An enzyme that links endocytosis to membrane recycling
07.12.2016 | National Centre for Biological Sciences
Transforming plant cells from generalists to specialists
07.12.2016 | Duke University
In recent years, lasers with ultrashort pulses (USP) down to the femtosecond range have become established on an industrial scale. They could advance some applications with the much-lauded “cold ablation” – if that meant they would then achieve more throughput. A new generation of process engineering that will address this issue in particular will be discussed at the “4th UKP Workshop – Ultrafast Laser Technology” in April 2017.
Even back in the 1990s, scientists were comparing materials processing with nanosecond, picosecond and femtosesecond pulses. The result was surprising:...
Have you ever wondered how you see the world? Vision is about photons of light, which are packets of energy, interacting with the atoms or molecules in what...
A multi-institutional research collaboration has created a novel approach for fabricating three-dimensional micro-optics through the shape-defined formation of porous silicon (PSi), with broad impacts in integrated optoelectronics, imaging, and photovoltaics.
Working with colleagues at Stanford and The Dow Chemical Company, researchers at the University of Illinois at Urbana-Champaign fabricated 3-D birefringent...
In experiments with magnetic atoms conducted at extremely low temperatures, scientists have demonstrated a unique phase of matter: The atoms form a new type of quantum liquid or quantum droplet state. These so called quantum droplets may preserve their form in absence of external confinement because of quantum effects. The joint team of experimental physicists from Innsbruck and theoretical physicists from Hannover report on their findings in the journal Physical Review X.
“Our Quantum droplets are in the gas phase but they still drop like a rock,” explains experimental physicist Francesca Ferlaino when talking about the...
The Max Planck Institute for Physics (MPP) is opening up a new research field. A workshop from November 21 - 22, 2016 will mark the start of activities for an innovative axion experiment. Axions are still only purely hypothetical particles. Their detection could solve two fundamental problems in particle physics: What dark matter consists of and why it has not yet been possible to directly observe a CP violation for the strong interaction.
The “MADMAX” project is the MPP’s commitment to axion research. Axions are so far only a theoretical prediction and are difficult to detect: on the one hand,...
16.11.2016 | Event News
01.11.2016 | Event News
14.10.2016 | Event News
07.12.2016 | Health and Medicine
07.12.2016 | Life Sciences
07.12.2016 | Health and Medicine