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; firstname.lastname@example.org
Emily Caldwell | Newswise Science News
Nanoparticle Exposure Can Awaken Dormant Viruses in the Lungs
16.01.2017 | Helmholtz Zentrum München - Deutsches Forschungszentrum für Gesundheit und Umwelt
Cholera bacteria infect more effectively with a simple twist of shape
13.01.2017 | Princeton University
Among the general public, solar thermal energy is currently associated with dark blue, rectangular collectors on building roofs. Technologies are needed for aesthetically high quality architecture which offer the architect more room for manoeuvre when it comes to low- and plus-energy buildings. With the “ArKol” project, researchers at Fraunhofer ISE together with partners are currently developing two façade collectors for solar thermal energy generation, which permit a high degree of design flexibility: a strip collector for opaque façade sections and a solar thermal blind for transparent sections. The current state of the two developments will be presented at the BAU 2017 trade fair.
As part of the “ArKol – development of architecturally highly integrated façade collectors with heat pipes” project, Fraunhofer ISE together with its partners...
At TU Wien, an alternative for resource intensive formwork for the construction of concrete domes was developed. It is now used in a test dome for the Austrian Federal Railways Infrastructure (ÖBB Infrastruktur).
Concrete shells are efficient structures, but not very resource efficient. The formwork for the construction of concrete domes alone requires a high amount of...
Many pathogens use certain sugar compounds from their host to help conceal themselves against the immune system. Scientists at the University of Bonn have now, in cooperation with researchers at the University of York in the United Kingdom, analyzed the dynamics of a bacterial molecule that is involved in this process. They demonstrate that the protein grabs onto the sugar molecule with a Pac Man-like chewing motion and holds it until it can be used. Their results could help design therapeutics that could make the protein poorer at grabbing and holding and hence compromise the pathogen in the host. The study has now been published in “Biophysical Journal”.
The cells of the mouth, nose and intestinal mucosa produce large quantities of a chemical called sialic acid. Many bacteria possess a special transport system...
UMD, NOAA collaboration demonstrates suitability of in-orbit datasets for weather satellite calibration
"Traffic and weather, together on the hour!" blasts your local radio station, while your smartphone knows the weather halfway across the world. A network of...
Fiber-reinforced plastics (FRP) are frequently used in the aeronautic and automobile industry. However, the repair of workpieces made of these composite materials is often less profitable than exchanging the part. In order to increase the lifetime of FRP parts and to make them more eco-efficient, the Laser Zentrum Hannover e.V. (LZH) and the Apodius GmbH want to combine a new measuring device for fiber layer orientation with an innovative laser-based repair process.
Defects in FRP pieces may be production or operation-related. Whether or not repair is cost-effective depends on the geometry of the defective area, the tools...
10.01.2017 | Event News
09.01.2017 | Event News
05.01.2017 | Event News
16.01.2017 | Trade Fair News
16.01.2017 | Automotive Engineering
16.01.2017 | Life Sciences