First step to untangle DNA: Supercoiled DNA captures gyrase like a lasso ropes cattle

Picture in your mind a traditional "landline" telephone with a coiled cord connecting the handset to the phone. The coiled telephone cord and the DNA double helix that stores the genetic material in every cell in the body have one thing in common; they both supercoil, or coil about themselves, and tangle in ways that can be difficult to undo. In the case of DNA, if this overwinding is not dealt with, essential processes such as copying DNA and cell division grind to a halt. Fortunately, cells have an ingenious solution to carefully regulate DNA supercoiling.

In this study published in the journal Science, researchers at Baylor College of Medicine, Université de Strasbourg, Université Paris Cité and collaborating institutions reveal how DNA gyrase resolves DNA entanglements. The findings not only provide novel insights into this fundamental biological mechanism but also have potential practical applications. Gyrases are biomedical targets for the treatment of bacterial infections and the similar human versions of the enzymes are targets for many anti-cancer drugs. Better understanding of how gyrases work at the molecular level can potentially improve clinical treatments.

Some DNA supercoiling is essential to make DNA accessible to allow the cell to read and make copies of the genetic information, but either too little or too much supercoiling is detrimental. For example, the act of copying and reading DNA overwinds it ahead of the enzymes that read and copy the genetic code, interrupting the process. It's long been known that DNA gyrase plays a role in untangling the overwinding, but the details were not clear.

DNA minicircles and advanced imaging techniques reveal first step to untangle DNA

"We typically picture DNA as the straight double helix structure, but inside cells, DNA exists in supercoiled loops. Understanding the molecular interactions between the supercoils and the enzymes that participate in DNA functions has been technically challenging, so we typically use linear DNA molecules instead of coiled DNA to study the interactions," said study author Dr. Lynn Zechiedrich, Kyle and Josephine Morrow Chair in Molecular Virology and Microbiology and professor of theVerna and Marrs McLean Department of Biochemistry and Molecular Pharmacology at Baylor College of Medicine. "One goal of our laboratory has been to study these interactions using a DNA structure that more closely mimics the actual supercoiled and looped DNA form present in living cells."

After years of work, the Zechiedrich lab has created small loops of supercoiled DNA. In essence, they took the familiar straight linear DNA double helix and twisted it in either direction once, twice, three times or more and connected the ends together to form a loop. Their previous study looking at the 3-D structures of the resulting supercoiled minicircles revealed that these loops form a variety of shapes that they hypothesized enzymes such as gyrase would recognize.

In the current study, their hypothesis was proven correct. The team of researchers combined their expertise to study the interactions of DNA gyrase with DNA minicircles using recent technology advances in electron cryomicroscopy, an imaging technique that produces high-resolution 3-D views of large molecules, and other technologies.

"My lab has long been interested in understanding how molecular nanomachines operate in the cell. We have been studying DNA gyrases, very large enzymes that regulate DNA supercoiling," said co-corresponding author Dr. Valérie Lamour, associate professor at the Institut de Génétique et de Biologie Moléculaire et Cellulaire, Université de Strasbourg. "Among other functions, supercoiling is the cell's way of confining about 2 meters (6.6 feet) of linear DNA into the microscopic nucleus of the cell."

As the DNA supercoils inside the nucleus, it twists and folds in different forms. Imagine twisting that telephone cord mentioned at the beginning, several times on itself. It will overwind and form a loop by crossing over DNA chains, tightening the structure.

"We found, just as we had hypothesized, that gyrase is attracted to the supercoiled minicircle and places itself in the inside of this supercoiled loop," said co-author, Dr. Jonathan Fogg, senior staff scientist of molecular virology and microbiology, and biochemistry and molecular pharmacology in the Zechiedrich lab.

"This is the first step of the mechanism that prompts the enzyme for resolving DNA entanglements," Lamour said.

"DNA gyrase, now surrounded by a tightly supercoiled loop, will cut one DNA helix in the loop, pass the other DNA helix through the cut in the other, and reseal the break, which relaxes the overwinding and eases the tangles, regulating DNA supercoiling to control DNA activity," Zechiedrich said. "Imagine watching the rodeo. Like roping cattle with a lasso, supercoiled looped DNA captures gyrase in the first step. Gyrase then cuts one double-helix of the DNA lasso and passes the other helix through the break to get free."

Co-corresponding author, Dr. Marc Nadal, professor at the École Normale in Paris confirmed the observation of the path of the DNA wrapped in the loop around gyrase using magnetic tweezers, a biophysical technique that allows to measure the deformation and fluctuations in the length of a single molecule of DNA. Observing a single molecule provides information that is often obscured when looking at thousands of molecules in traditional so-called "ensemble" experiments in a test tube.

Interestingly, the "DNA strand inversion model" for gyrase activity was proposed in 1979 by Drs. Patrick O. Brown and the late Nicholas R. Cozzarelli, also in a Science paper, well before researchers had access to supercoiled minicircles or the 3-D molecular structure of the enzyme. "It's especially meaningful to me that 45 years later, we finally provide experimental evidence supporting their hypothesis because Nick was my postdoctoral mentor," Zechiedrich said.

"This work opens a myriad of perspectives to study the mechanism of this conserved class of enzymes, which are of great clinical value," Lamour said.

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Modeling the origins of life: New evidence for an 'RNA World'

Charles Darwin described evolution as "descent with modification." Genetic information in the form of DNA sequences is copied and passed down from one generation to the next. But this process must also be somewhat flexible, allowing slight variations of genes to arise over time and introduce new traits into the population.

But how did all of this begin? In the origins of life, long before cells and proteins and DNA, could a similar sort of evolution have taken place on a simpler scale? Scientists in the 1960s, including Salk Fellow Leslie Orgel, proposed that life began with the "RNA World," a hypothetical era in which small, stringy RNA molecules ruled the early Earth and established the dynamics of Darwinian evolution.

New research at the Salk Institute now provides fresh insights on the origins of life, presenting compelling evidence supporting the RNA World hypothesis. The study, published in Proceedings of the National Academy of Sciences (PNAS) on March 4, 2024, unveils an RNA enzyme that can make accurate copies of other functional RNA strands, while also allowing new variants of the molecule to emerge over time. These remarkable capabilities suggest the earliest forms of evolution may have occurred on a molecular scale in RNA.

The findings also bring scientists one step closer to re-creating RNA-based life in the laboratory. By modeling these primitive environments in the lab, scientists can directly test hypotheses about how life may have started on Earth, or even other planets.

"We're chasing the dawn of evolution," says senior author and Salk President Gerald Joyce. "By revealing these novel capabilities of RNA, we're uncovering the potential origins of life itself, and how simple molecules could have paved the way for the complexity and diversity of life we see today."

Scientists can use DNA to trace the history of evolution from modern plants and animals all the way back to the earliest single-celled organisms. But what came before that remains unclear. Double-stranded DNA helices are great for storing genetic information. Many of those genes ultimately code for proteins -- complex molecular machines that carry out all sorts of functions to keep cells alive. What makes RNA unique is that these molecules can do a bit of both. They're made of extended nucleotide sequences, similar to DNA, but they can also act as enzymes to facilitate reactions, much like proteins. So, is it possible that RNA served as the precursor to life as we know it?

Scientists like Joyce have been exploring this idea for years, with a particular focus on RNA polymerase ribozymes -- RNA molecules that can make copies of other RNA strands. Over the last decade, Joyce and his team have been developing RNA polymerase ribozymes in the lab, using a form of directed evolution to produce new versions capable of replicating larger molecules. But most have come with a fatal flaw: they aren't able to copy the sequences with a high enough accuracy. Over many generations, so many errors are introduced into the sequence that the resulting RNA strands no longer resemble the original sequence and have lost their function entirely.

Until now. The latest RNA polymerase ribozyme developed in the lab includes a number of crucial mutations that allow it to copy a strand of RNA with much higher accuracy.

In these experiments, the RNA strand being copied is a "hammerhead," a small molecule that cleaves other RNA molecules into pieces. The researchers were surprised to find that not only did the RNA polymerase ribozyme accurately replicate functional hammerheads, but over time, new variations of the hammerheads began to emerge. These new variants performed similarly, but their mutations made them easier to replicate, which increased their evolutionary fitness and led them to eventually dominate the lab's hammerhead population.

"We've long wondered how simple life was at its beginning and when it gained the ability to start improving itself," says first author Nikolaos Papastavrou, a research associate in Joyce's lab. "This study suggests the dawn of evolution could have been very early and very simple. Something at the level of individual molecules could sustain Darwinian evolution, and that might have been the spark that allowed life to become more complex, going from molecules to cells to multicellular organisms."

The findings highlight the critical importance of replication fidelity in making evolution possible. The RNA polymerase's copying accuracy must exceed a critical threshold to maintain heritable information over multiple generations, and this threshold would have risen as the evolving RNAs increased in size and complexity.

Joyce's team is re-creating this processin laboratory test tubes, applying increasing selective pressure on the system to produce better-performing polymerases, with the goal of one day producing an RNA polymerase that can replicate itself. This would mark the beginnings of autonomous RNA life in the laboratory, which the researchers say could be accomplished within the next decade.

The scientists are also interested in what else might occur once this mini "RNA World" has gained more autonomy.

"We've seen that selection pressure can improve RNAs with an existing function, but if we let the system evolve for longer with larger populations of RNA molecules, can new functions be invented?" says co-author David Horning, a staff scientist in Joyce's lab. "We're excited to answer how early life could ratchet up its own complexity, using the tools developed here at Salk."

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Researchers model 'link' between improved photosynthesis and increased yield

A team from the University of Illinois has modeled improving photosynthesis through enzyme modification and simulated soybean growth with realistic climate conditions, determining to what extent the improvements in photosynthesis could result in increased yields.

"There's a complex relationship between photosynthesis improvement and actual yield, having higher photosynthesis doesn't necessarily mean you have higher yield. The yield return is highly impacted by seasonal climate conditions" said Yufeng He, a postdoctoral researcher at Illinois, who led this work for a research project called Realizing Increased Photosynthetic Efficiency (RIPE). "This study has created a bridge that links the missing part between photosynthesis improvements and higher yields at field scale."

RIPE, which is led by Illinois, is engineering crops to be more productive by improving photosynthesis, the natural process all plants use to convert sunlight into energy and yields. This RIPE research was supported by the Bill & Melinda Gates Foundation, Foundation for Food & Agriculture Research, and U.K. Foreign, Commonwealth & Development Office.

He and his colleagues in the Matthews Research Group used the BioCro modeling framework to simulate soybeans in Illinois fields under normal and elevated CO2 conditions, paying specific attention to two important parameters that affect the plant canopy's photosynthetic process; Jmax and Vcmax. They wanted to determine the effect of boosting these photosynthetic processes at the canopy level, rather than just at the leaf level, and determine if the effects could lead to higher yields under a range of climate conditions.

The team found that the overall returns in plant photosynthesis and pod biomass (yields) were affected when plants were simulated in a high CO2 environment. They also found that correlations between increased photosynthesis and increased yield were dependent on the climate conditions at different stages of soybean growth. Their findings were recently published in Field Crops Research.

"There has been evidence showing that photosynthesis can be improved by modifying certain enzymes, but most of these studies were either done only looking at the leaf-scale impacts or the impacts from a limited number of field trials and seasonal climate conditions," said Megan Matthews, Assistant Professor in the Department of Civil and Environmental Engineering at Illinois and Principal Investigator on the research. "We studied the impacts of seasonal climate conditions at the field level on the improvements of photosynthesis. Using realistic climate inputs to run our models and show how those improvements would vary with different climates."

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Back to the future of photosynthesis

The central biocatalyst in photosynthesis, Rubisco, is the most abundant enzyme on earth. By reconstructing billion-year-old enzymes, a team of Max Planck researchers has deciphered one of the key adaptations of early photosynthesis. Their results not only provide insights into the evolution of modern photosynthesis but also offer new impulses for improving it.

Present day life fully depends on photosynthetic organisms like plants and algae that capture and convert CO2. At the heart of these processes lies an enzyme called Rubisco that captures more than 400 billion tons CO2 annually. Organisms alive today make staggering amounts of it: the mass of Rubisco on our planet outweighs that of all humans. In order to assume such a dominant role in the global carbon cycle, Rubisco had to adapt constantly to changing environmental conditions.

Using a combination of computational and synthetic approaches, a team from the Max Planck Institute for Terrestrial Microbiology in Marburg, Germany, in collaboration with the University of Singapore has now successfully resurrected and studied billion-year-old enzymes in the lab. In this process, which they describe as "molecular paleontology," the researchers found that instead of direct mutations in the active center, an entirely new component prepared photosynthesis to adapt to rising oxygen levels.

Rubisco's early confusion

Rubisco is ancient: it emerged approximately four billion years ago in primordial metabolism prior to the presence of oxygen on earth. However, with the invention of oxygen-producing photosynthesis and rise of oxygen in the atmosphere, the enzyme started catalyzing an undesired reaction, in which it mistakes O2 for CO2 and produces metabolites that are toxic to the cell. This confused substrate scope still scars Rubiscos to date and limits photosynthetic efficiency. Even though Rubiscos that evolved in oxygen-containing environments became more specific for CO2 over time, none of them could get completely rid of the oxygen capturing reaction.

The molecular determinants of increased CO2 specificity in Rubisco remain largely unknown. However, they are of great interest to researchers aiming to improve photosynthesis. Interestingly, those Rubiscos that show increased CO2 specificity recruited a novel protein component of unknown function. This component was suspected to be involved in increasing CO2 specificity, however, the true reason for its emergence remained difficult to determine because it already evolved billions of years ago.

Studying evolution by resurrecting ancient proteins in the lab

To understand this key event in the evolution of more specific Rubiscos, collaborators at the Max Planck Institute for Terrestrial Microbiology in Marburg and Nanyang Technological University in Singapore used a statistical algorithm to recreate forms of Rubiscos that existed billions of years ago, before oxygen levels began to rise. The team led by Max Planck researchers Tobias Erb and Georg Hochberg resurrected these ancient proteins in the lab to study their properties. In particular, the scientists wondered whether Rubisco's new component had anything to do with the evolution of higher specificity.

The answer was surprising, as doctoral researcher Luca Schulz explains: "We expected the new component to somehow directly exclude oxygen from Rubisco catalytic center. That is not what happened. Instead, this new subunit seems to act as a modulator for evolution: recruitment of the subunit changed the effect that subsequent mutations had on Rubisco's catalytic subunit. Previously inconsequential mutations suddenly had a huge effect on specificity when this new component was present. It seems that having this new subunit completely changed Rubisco's evolutionary potential."

An enzyme's addiction to its new subunit

This function as an "evolutionary modulator" also explains another mysterious aspect of the new protein component: Rubiscos that incorporated it are completely dependent on it, even though other forms of Rubisco can function perfectly well without. The same modulating effect explains why: When bound to this small protein component, Rubisco become tolerant to mutations that would otherwise be catastrophically detrimental. With the accumulation of such mutations, Rubisco effectively became addicted to its new subunit.

Altogether, the findings finally explain the reason why Rubisco kept this new protein component around ever since it encountered it. Max Planck Research Group Leader Georg Hochberg explains: "The fact that this connection was not understood until now highlights the importance of evolutionary analysis for understanding the biochemistry that drives life around us. The history of biomolecules like Rubisco can teach us so much about why they are the way they are today. And there are still so many biochemical phenomena whose evolutionary history we really have no idea about. So it's a very exciting time to be an evolutionary biochemist: almost the entire molecular history of the cell is still waiting to be discovered."

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How a soil microbe could rev up artificial photosynthesis

Plants rely on a process called carbon fixation -- turning carbon dioxide from the air into carbon-rich biomolecules - for their very existence. That's the whole point of photosynthesis, and a cornerstone of the vast interlocking system that cycles carbon through plants, animals, microbes and the atmosphere to sustain life on Earth.

But the carbon fixing champs are not plants, but soil bacteria. Some bacterial enzymes carry out a key step in carbon fixation 20 times faster than plant enzymes do, and figuring out how they do this could help scientists develop forms of artificial photosynthesis to convert the greenhouse gas into fuels, fertilizers, antibiotics and other products.

Now a team of researchers from the Department of Energy's SLAC National Accelerator Laboratory, Stanford University, Max Planck Institute for Terrestrial Microbiology in Germany, DOE's Joint Genome Institute (JGI) and the University of Concepción in Chile has discovered how a bacterial enzyme -- a molecular machine that facilitates chemical reactions -- revs up to perform this feat.

Rather than grabbing carbon dioxide molecules and attaching them to biomolecules one at a time, they found, this enzyme consists of pairs of molecules that work in sync, like the hands of a juggler who simultaneously tosses and catches balls, to get the job done faster. One member of each enzyme pair opens wide to catch a set of reaction ingredients while the other closes over its captured ingredients and carries out the carbon-fixing reaction; then, they switch roles in a continual cycle.

A single spot of molecular "glue" holds each pair of enzymatic hands together so they can alternate opening and closing in a coordinated way, the team discovered, while a twisting motion helps hustle ingredients and finished products in and out of the pockets where the reactions take place. When both glue and twist are present, the carbon-fixing reaction goes 100 times faster than without them.

"This bacterial enzyme is the most efficient carbon fixer that we know of, and we came up with a neat explanation of what it can do," said Soichi Wakatsuki, a professor at SLAC and Stanford and one of the senior leaders of the study, which was published in ACS Central Science this week.

"Some of the enzymes in this family act slowly but in a very specific way to produce just one product," he said. "Others are much faster and can craft chemical building blocks for all sorts of products. Now that we know the mechanism, we can engineer enzymes that combine the best features of both approaches and do a very fast job with all sorts of starting materials."

Improving on nature

The enzyme the team studied is part of a family called enoyl-CoA carboxylases/reductases, or ECRs. It comes from soil bacteria called Kitasatospora setae, which in addition to their carbon-fixing skills can also produce antibiotics.

Wakatsuki heard about this enzyme family half a dozen years ago from Tobias Erb of the Max Planck Institute for Terrestrial Microbiology in Germany and Yasuo Yoshikuni of JGI. Erb's research team had been working to develop bioreactors for artificial photosynthesis to convert carbon dioxide (CO2) from the atmosphere into all sorts of products.

As important as photosynthesis is to life on Earth, Erb said, it isn't very efficient. Like all things shaped by evolution over the eons, it's only as good as it needs to be, the result of slowly building on previous developments but never inventing something entirely new from scratch.

What's more, he said, the step in natural photosynthesis that fixes CO2 from the air, which relies on an enzyme called Rubisco, is a bottleneck that bogs the whole chain of photosynthetic reactions down. So using speedy ECR enzymes to carry out this step, and engineering them to go even faster, could bring a big boost in efficiency.

"We aren't trying to make a carbon copy of photosynthesis," Erb explained. "We want to design a process that's much more efficient by using our understanding of engineering to rebuild the concepts of nature. This 'photosynthesis 2.0' could take place in living or synthetic systems such as artificial chloroplasts -- droplets of water suspended in oil."

Portraits of an enzyme

Wakatsuki and his group had been investigating a related system, nitrogen fixation, which converts nitrogen gas from the atmosphere into compounds that living things need. Intrigued by the question of why ECR enzymes were so fast, he started collaborating with Erb's group to find answers.

Hasan DeMirci, a research associate in Wakatsuki's group who is now an assistant professor at Koc University and investigator with the Stanford PULSE Institute, led the effort at SLAC with help from half a dozen SLAC summer interns he supervised. "We train six or seven of them every year, and they were fearless," he said. "They came with open minds, ready to learn, and they did amazing things."

The SLAC team made samples of the ECR enzyme and crystallized them for examination with X-rays at the Advanced Photon Source at DOE's Argonne National Laboratory. The X-rays revealed the molecular structure of the enzyme -- the arrangement of its atomic scaffolding -- both on its own and when attached to a small helper molecule that facilitates its work.

Further X-ray studies at SLAC's Stanford Synchrotron Radiation Lightsource (SSRL) showed how the enzyme's structure shifted when it attached to a substrate, a kind of molecular workbench that assembles ingredients for the carbon fixing reaction and spurs the reaction along.

Finally, a team of researchers from SLAC's Linac Coherent Light Source (LCLS) carried out more detailed studies of the enzyme and its substrate at Japan's SACLA X-ray free-electron laser. The choice of an X-ray laser was important because it allowed them to study the enzyme's behavior at room temperature -- closer to its natural environment -- with almost no radiation damage.

Meanwhile, Erb's group in Germany and Associate Professor Esteban Vo?hringer-Martinez's group at the University of Concepción in Chile carried out detailed biochemical studies and extensive dynamic simulations to make sense of the structural data collected by Wakatsuki and his team.

The simulations revealed that the opening and closing of the enzyme's two parts don't just involve molecular glue, but also twisting motions around the central axis of each enzyme pair, Wakatsuki said.

"This twist is almost like a rachet that can push a finished product out or pull a new set of ingredients into the pocket where the reaction takes place," he said. Together, the twisting and synchronization of the enzyme pairs allow them to fix carbon 100 times a second.

The ECR enzyme family also includes a more versatile branch that can interact with many different kinds of biomolecules to produce a variety of products. But since they aren't held together by molecular glue, they can't coordinate their movements and therefore operate much more slowly.

"If we can increase the rate of those sophisticated reactions to make new biomolecules," Wakatsuki said, "that would be a significant jump in the field."

From static shots to fluid movies

So far the experiments have produced static snapshots of the enzyme, the reaction ingredients and the final products in various configurations.

"Our dream experiment," Wakatsuki said, "would be to combine all the ingredients as they flow into the path of the X-ray laser beam so we could watch the reaction take place in real time."

The team actually tried that at SACLA, he said, but it didn't work. "The CO2 molecules are really small, and they move so fast that it's hard to catch the moment when they attach to the substrate," he said. "Plus the X-ray laser beam is so strong that we couldn't keep the ingredients in it long enough for the reaction to take place. When we pressed hard to do this, we managed to break the crystals."

An upcoming high-energy upgrade to LCLS will likely solve that problem, he added, with pulses that arrive much more frequently -- a million times per second -- and can be individually adjusted to the ideal strength for each sample.

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Scientists resurrect ancient enzymes to improve photosynthesis

A Cornell University study describes a breakthrough in the quest to improve photosynthesis in certain crops, a step toward adapting plants to rapid climate changes and increasing yields to feed a projected 9 billion people by 2050.

The study, "Improving the Efficiency of Rubisco by Resurrecting Its Ancestors in the Family Solanaceae," published April 15 in Science Advances. The senior author is Maureen Hanson, the Liberty Hyde Bailey Professor of Plant Molecular Biology in the College of Agriculture and Life Sciences. First author Myat Lin is a postdoctoral research associate in Hanson's lab.

The authors developed a computational technique to predict favorable gene sequences that make Rubisco, a key plant enzyme for photosynthesis. The technique allowed the scientists to identify promising candidate enzymes that could be engineered into modern crops and, ultimately, make photosynthesis more efficient and increase crop yields.

Their method relied on evolutionary history, where the researchers predicted Rubisco genes from 20-30 million years ago, when Earth's carbon dioxide (CO2) levels were higher than they are today and the Rubisco enzymes in plants were adapted to those levels.

By resurrecting ancient Rubisco, early results show promise for developing faster, more efficient Rubisco enzymes to incorporate into crops and help them adapt to hot, dry future conditions, as human activities are increasing heat-trapping CO2 gas concentrations in Earth's atmosphere.

The study describes predictions of 98 Rubisco enzymes at key moments in the evolutionary history of plants in the Solanaceae family, which include tomato, pepper, potato, eggplant and tobacco. Researchers use tobacco as the experimental model for their studies of Rubisco.

"We were able to identify predicted ancestral enzymes that do have superior qualities compared to current-day enzymes," Hanson said. Lin developed the new technique for identifying predicted ancient Rubisco enzymes.

Scientists have known that they can increase crop yields by accelerating photosynthesis, where plants convert CO2, water and light into oxygen and sugars that plants use for energy and for building new tissues.

For many years, researchers have focused on Rubisco, a slow enzyme that pulls (or fixes) carbon from CO2 to create sugars. Aside from being slow, Rubisco also sometimes catalyzes a reaction with oxygen in the air; by so doing, it creates a toxic byproduct, wastes energy and makes photosynthesis inefficient.

Hanson's lab had previously tried to use Rubisco from cyanobacteria (blue-green algae), which is faster but also reacts readily with oxygen, forcing the researchers to try to create micro-compartments to protect the enzyme from oxygen, with mixed results. Other researchers have tried to engineer more optimal Rubisco by making changes in the enzyme's amino acids, though little was known about which changes would lead to desired results.

In this study, Lin reconstructed a phylogeny -- a tree-like diagram showing evolutionary relatedness among groups of organisms -- of Rubisco, using Solanaceae plants.

"By getting a lot of [genetic] sequences of Rubisco in existing plants, a phylogenetic tree could be constructed to figure out which Rubiscos likely existed 20 to 30 million years ago," Hanson said.

The advantage of identifying potential ancient Rubisco sequences is that carbon dioxide levels were possibly as high as 500 to 800 parts per million (ppm) in the atmosphere 25 million to 50 million years ago. Today, heat-trapping CO2 levels are rising sharply due to many human activities, with current measurements at around 420 ppm, after staying relatively constant under 300 ppm for hundreds of millennia until the 1950s.

Lin, Hanson and colleagues then used an experimental system developed for tobacco in Hanson's lab, and described in a 2020 Nature Plants paper, which employs E. coli bacteria to test in a single day the efficacy of different versions of Rubisco. Similar tests done in plants take months to verify.

The team found that ancient Rubisco enzymes predicted from modern-day Solanaceae plants showed real promise for being more efficient.

"For the next step, we want to replace the genes for the existing Rubisco enzyme in tobacco with these ancestral sequences using CRISPR [gene-editing] technology, and then measure how it affects the production of biomass," Hanson said. "We certainly hope that our experiments will show that by adapting Rubisco to present day conditions, we will have plants that will give greater yields."

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Scaling laws in enzymes may help predict life ‘as we don’t know it'

The only references we have for "life" are the forms we know on Earth. Astrobiologists suspect that the search for alien life, and even for the origins of life on Earth, may require a broader scope. A NASA-funded team of researchers is developing tools to predict the features of life as we don't know it. In a new study published in the Proceedings of the National Academy of Sciences, the team identifies universal patterns in the chemistry of life that do not appear to depend on specific molecules.

"We want to have new tools for identifying and even predicting features of life as we don't know it," says Santa Fe Institute External Professor Sara Imari Walker (Arizona State University), a co-author on the paper. "To do so, we are aiming to identify the universal laws that should apply to any biochemical system. This includes developing quantitative theory for the origins of life, and using theory and statistics to guide our search for life on other planets."

On Earth, life emerges from the interplay of hundreds of chemical compounds and reactions. Some of these compounds and reactions are found universally across Earth's organisms. Using the Integrated Microbial Genomes and Microbiomes database, the team investigated the enzymes -- the functional drivers of biochemistry -- found in bacteria, archaea, and eukarya to reveal a new kind of biochemical universality.

Enzymes can be categorized into a taxonomy of broad functional classes -- groups designated by what they do, from using water molecules to break chemical bonds (hydrolases) to rearranging molecular structures (isomerases) to joining large molecules together (ligases). The team compared how the abundance of enzymes in each of these functional categories changed in relation to the overall abundance of enzymes in an organism. They discovered various scaling laws -- almost algorithmic relationships -- between the number of enzymes in different enzyme classes and the size of an organism's genome. They also found that these laws don't depend on the particularenzymes in those classes.

"Here we find that you get these scaling relationships without needing to conserve exact membership. You need a certain number of transferases, but not particular transferases," says SFI Professor Chris Kempes, a co-author on the paper. "There are a lot 'synonyms,' and those synonyms scale in systematic ways."

On Earth, organisms use DNA and, through RNA, create proteins. But will the macromolecules of DNA, RNA, and proteins help us identify life across the universe, understand the origins of life on Earth, or develop synthetic biology? "As a team, we think that's not likely," says Kempes. The functions those macromolecules serve, however, and the metabolic scaling relationships observed in organic, Earth-based life, just might be. "Even if life elsewhere used really different molecules, these sort of functional categories and scaling laws might be conserved throughout the universe," says Kempes.

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Do genetics control who our friends are? It seems so with mice

Have you ever met someone you instantly liked, or at other times, someone who you knew immediately that you did not want to be friends with, although you did not know why?

Popular author Malcolm Gladwell examined this phenomenon in his best-selling book, Blink. In his book, he noted that an "unconscious" part of the brain enables us to process information spontaneously, when, for example, meeting someone for the first time, interviewing someone for a job, or faced with making a decision quickly under stress.

Now, a new study from the University of Maryland School of Medicine (UMSOM) suggests that there may be a biological basis behind this instantaneous compatibility reaction. A team of researchers showed that variations of an enzyme found in a part of the brain that regulates mood and motivation seems to control which mice want to socially interact with other mice -- with the genetically similar mice preferring each other.

The UMSOM researchers, led by Michy Kelly, PhD, Associate Professor of Anatomy and Neurobiology, say their findings may indicate that similar factors could contribute to the social choices people make. Understanding what factors drive these social preferences may help us to better recognize what goes awry in diseases associated with social withdrawal, such as schizophrenia or autism, so that better therapies can be developed.

The study was published on July 28 in Molecular Psychiatry, a Nature publication.

"We imagine that this is only the first among many biomarkers of compatibility in the brain that may control social preferences," said Dr. Kelly. "Imagine the possibilities of truly understanding the factors behind human compatibility. You could better match relationships to reduce heartache and divorce rates, or better match patients and doctors to advance the quality of healthcare, as studies have shown compatibility can improve health outcomes."

A succession of unlikely events and circumstances over the years eventually culminated in this research project, according to Dr. Kelly.

While she was working at a pharmaceutical company, a group of bone researchers asked Dr. Kelly to characterize the behavior of one of their mutant mice that was missing the PDE11 protein. She observed that these mice without PDE11 withdrew socially, so she knew that PDE11 had to be in the brain. She remembered a study that used a mouse model of schizophrenia in which the researchers damaged the brain's hippocampus leading to antisocial behavior. She then looked at this part of the brain in healthy mice and found where the PDE11 protein was hiding.

Later, as a faculty member at University of South Carolina, she continued studying the social behavior of mutant mice in terms of their social reactions to scent. In the lab, researchers took wooden beads rubbed all over with pungent, airborne pheromones from one group of mice, and placed them in an enclosure with a second group. A mouse presented with one bead from a familiar friend and another from a new stranger mouse would typically spend more time investigating the bead with the stranger's scent on it. When researchers looked at the PDE11 mutant's preferences, they favored the stranger's scent one hour or one week after meeting their friend, but one day after meeting -- considered recent long-term memory for a mouse -- their social memory seemed fuzzy, and they did not differentiate between a friend and a stranger. To the researchers this meant, the mice's short and long-term social memory worked fine, but there was a problem coding the information into recent long-term memory -- the time between short and long-term memory. Given more time, they would eventually recover that memory.

A student working in the laboratory offhandedly remarked that he noticed children with autism prefer to interact with others that have autism. So, Dr. Kelly decided they should test to see if the PDE11 mutants and normal mice had a preference with whom they interacted. The researchers found that PDE11 mutants preferred being around other PDE11 mutants over the normal mice, while normal mice also preferred their own genetic type. This discovery held true even when researchers tested other laboratory mouse strains. When they tested another genetic variant of PDE11 with a single change in the DNA code, mice with that genetic variation preferred other mice with the same variant over any others.

"So, what is it that the mice are sensing that determines their friend preferences?" said Dr. Kelly. "We eliminated smell and body movements as contributing factors, but we still have some other ideas to test."

"What this team has done is to establish a paradigm by which researchers can identify the social underpinnings of friendship in animal models," said E. Albert Reece, MD, PhD, MBA, Executive Vice President for Medical Affairs, UM Baltimore, and the John Z. and Akiko K. Bowers Distinguished Professor and Dean, University of Maryland School of Medicine. "This very important finding is just the start, but hopefully will lead to exciting new avenues of biological or social treatments for diseases like schizophrenia or age-related cognitive decline in which severe social avoidance and isolation can reduce a person's quality of life."

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Mutated enzyme weakens connection between brain cells that help control movement

In one type of a rare, inherited genetic disorder that affects control of body movement, scientists have found a mutation in an enzyme impairs communication between neurons and what should be the inherent ability to pick up our pace when we need to run, instead of walk, across the street.

The disorder is spinocerebellar ataxia, or SCA, a neurodegenerative condition resulting from different genetic mutations whose debilitating bottom line can include ataxia -- loss of control of body movement -- and atrophy of the cerebellum, a small part of the brain jam packed with neurons, which coordinates movement and balance, says Dr. Ferenc Deak, neuroscientist at the Medical College of Georgia at Augusta University.

The enzyme is ELOVL4, which produces very long chain fatty acids, and its mutation is known to cause the specific SCA type 34. Animal models with this SCA type have problems with motor control by age two months, and scientists from MCG and the University of Oklahoma Health Sciences Center wanted to know precisely why.

"We found a dramatically diminished synaptic response. The information was to go faster, go faster and they never really got the message," Deak, co-corresponding author of the study in the journal Molecular Neurobiology, says of these communication connections between neurons. "They were transmitting the signal, but when they had to adjust their synaptic connection to coordinate the different movement, that did not happen in the mutant knock-in rat," he says of the SCA34 model generated using the gene editing technique CRISPR cas9.

Despite the different gene mutations that are causative in SCA, a common bottom line appears to be altered output of the cerebellum and an impact on Purkinje cells, big brain cells in the cerebellum, which can receive about 100 times the input of usual neurons. The big cells also exclusively inhibit communication, so they shut down signals that would interfere with something like a muscle being activated. Loss of these key cells is clear in many forms of SCA, Deak says.

Much like an air traffic controller at a busy airport, these big brain cells obviously monitor a lot of different input simultaneously, and they are the only neuron sending out messages from that part of the brain.

Purkinje cells get a lot of their input from granule cells, one of the smallest neurons in the brain but largest in number. Both cell types express a lot of ELOVL4 and also depend on the enzyme, Deak says. ELOVL4 was known to be important to the communication between these and other cells, but why remained elusive.

The new studies found mutation of ELOVL4 resulted in significant reduction of the ability of synapses that bring messages to and away from Purkinje cells to strengthen their signaling, which is essential in this case to coordinating movement, so you could speed up your pace if needed or move your hands wildly about on command.

Their findings point to the essential role of ELOVL4 in motor function and synaptic plasticity, Deak says.

They also suggest that patients with SCA34 have an impairment and asynchrony in the communication between key neurons in the cerebellum well before their brain shows clear signs of degeneration.

Deak notes that over time, the impaired responses between these constantly communicating cells may lead to the degeneration of the cerebellum often found in patients when they first go to their doctor with complaints about problems with walking, talking and other movement.

But in their model of SCA34, the structure of the cerebellum looked normal up to six months of age, even though the animal models clearly had the expected motor deficits, the scientists report.

They found the synapses also were intact and functioning at a basic level that enables the rats to, for example, walk normally, but in these knock-in rats the usual plasticity or flexibility was lacking. Rather synapses in the mutant couldn't increase signaling and make that transition.

ELOVL4 can make both saturated and unsaturated very long chain fatty acids -- dubbed "long" because of the large number of carbon atoms they contain -- depending on which tissue the enzyme is in. In the cerebellum, it enables Purkinje and granule cells to make saturated very long chain fatty acids, which were known to be important to synaptic function, Deak says. But exactly how they are important was an unknown.

The scientists think the weakened synaptic responsiveness they found is a quantity problem: the mutated enzyme makes about 70% of the usual amount of very long chain fatty acids, which appears to be the threshold for gait problems. If the cells produced none, it would result in excessive seizures and death as Deak has seen in other models.

Their current research includes finding ways to deliver more saturated very long chain fatty acids to the brain. The scientists have a patent pending on one way to make this maneuver, which is made tougher by the fact that when you produce saturated very long chain fatty acids they have the consistency of candle wax, Deak says, which the rats don't even digest, just poop out.

Very long chain fatty acids are essential to life but their exact roles are mostly elusive, the scientists say.

"What we know from our work is that they are a very important component for certain cell membranes," Deak says, like the membranes of some excitatory and inhibitory neurons as well as skin cells. In fact, the scientists have shown that when ELOVL4 is missing in the skin, body fluids seep through the skin, our largest natural barrier. In generating other ELOVL4 mutant mice models they had to overexpress ELOVL4 specifically in the skin to enable survival, Deak said.

Deak's research has shown that these saturated very long chain fatty acids also like to accumulate and strengthen vesicles, tiny traveling compartments that can move about inside cells, so they are better able to get to their destination before they fuse with a cell membrane. Fusing is necessary for neurotransmission -- one of the things vesicles in the brain transport is chemical messengers called neurotransmitters -- but unregulated fusion is bad.

The scientists documented its impact when they found that mice with two mutant copies of ELOVL4 died of seizures. While they were making these findings in the laboratory, there were reports out of Saudi Arabia about children having the same mutations and issues, he says. In fact, it was Deak's research interest in seizures that prompted his pursuit of better understanding the roles of very long chain fatty acids. He suspects they have a role in his other interest as well: the aging brain and Alzheimer's.

A team led by Dr. Martin-Paul Agbaga created the "knock-in" rat model of the human condition SCA34, which has been identified in one French-Canadian family and three Japanese families. In these individuals, skin problems can surface from shortly after birth to adolescence, and movement problems and typically progressive movement issues start surfacing in their 30s.

Agbaga, co-corresponding author of the new paper and one of Deak's longtime collaborators, is a vision scientist and cell biologist at the University of Oklahoma Health Sciences Center Department of Ophthalmology and Dean McGee Eye Institute.

Dr. Robert E. Anderson, professor of vision research at the Dean McGee Eye Institute, founder of the ELOVL4 research group in Oklahoma, and a worldwide leader of research on lipid pathophysiology in the retina, is a coauthor on the paper. Deak came to MCG from the University of Oklahoma Health Sciences Center last year.

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New study details enzyme that allows coronavirus to resist antiviral medications

The coronavirus that causes COVID-19 has demonstrated a stubborn ability to resist most nucleoside antiviral treatments, but a new study led by an Iowa State University scientist could help to overcome the virus's defenses.

The study, published recently in the peer-reviewed journal Science, details the structure of a critical enzyme present in SARS-CoV-2, the coronavirus that causes COVID-19. This enzyme, known as the proofreading exoribonuclease (or ExoN), removes nucleoside antiviral medications from the virus's RNA, rendering most nucleoside analogs-based antiviral treatments ineffective. The new study presents the atomic structures of the ExoN enzyme, which could lead to the development of new methods for deactivating the enzyme and opening the door to better treatments for patients suffering from COVID-19.

"If we could find a way to inhibit this enzyme, maybe we can achieve better results to kill the virus with existing nucleoside antiviral treatments. Understanding this structure and the molecular details of how ExoN works can help guide further development of antivirals," said Yang Yang, lead author of the study and assistant professor in the Roy J. Carver Department of Biochemistry, Biophysics and Molecular Biology at Iowa State University.

SARS-CoV-2 is an RNA virus, which means its genetic material is composed of ribonucleic acid. When the virus replicates, it must synthesize RNA. But the virus's genome is unusually large when compared to other RNA viruses, which creates a relatively high likelihood that errors arise during RNA synthesis. These errors take the form of mismatched nucleotides, and too many errors can prevent the virus from propagating.

But the ExoN enzyme acts as a proofreader, recognizing mismatches in the virus RNA and correcting errors that occur during RNA synthesis, Yang said. The enzyme is present only in coronaviruses and a few other closely related virus families, he said.

The same process that eliminates replication errors also eliminates antiviral agents delivered by the treatments commonly used to fight other RNA viruses, such as HIV, HCV and Ebola virus, which partially explains why SARS-CoV-2 has proven so difficult to treat, Yang said.

But Yang and his colleagues utilized cryogenic electron microscopy, a technique in which samples are flash cooled to cryogenic temperatures in vitreous ice to preserve their native structures, to detail the structure of the enzyme. Understanding that structure could allow for the development of molecules that bind to the enzyme and disable it. Yang said that's the next step for his laboratory and his colleagues. Finding such a molecule could make the virus more susceptible to newly developed antivirals, Yang said. Or, it could allow for the optimization of current antivirals, such as Remdesivir.

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Superficial relationship: Enzymes protect the skin by ignoring microbes and viruses

The human body is constantly exposed to various environmental actors, from viruses to bacteria to fungi, but most of these microbial organisms provoke little or no response from our skin, which is charged with monitoring and protecting from external dangers.

Until now, researchers weren't quite sure how that happened -- and why our skin wasn't constantly alarmed and inflamed.

In a study published May 21, 2021 in Science Immunology, scientists at University of California San Diego School of Medicine identify and describe two enzymes responsible for protecting our skin and body's overall health from countless potential microbial intruders. These enzymes, called histone deacetylases (HDACs), inhibit the body's inflammatory response in the skin.

"We have figured out why we tolerate certain microbes living on our skin, while the same bacteria would make us very sick if exposed elsewhere in the body," said Richard Gallo, MD, PhD, Ima Gigli Distinguished Professor of Dermatology and chair of the Department of Dermatology at UC San Diego School of Medicine. "In our research, we identified enzymes that act on the chromosome of specific skin cells that provide immune tolerance by the skin.

"Without these enzymes telling our cells to ignore certain bacteria, we'd have a constant rash on our skin."

Gallo and colleagues say the potential mechanism for how the environment can interact and alter cell function is through epigenetic control of gene expression. Within the skin cells, proteins called toll-like receptors (TLRs) allow the cells to sense their surroundings and potential dangers.

In most organs, TLRs act as a warning system that triggers an inflammatory response to threats. But in skin cells, the two identified HDAC enzymes, HDAC8 and HDAC9, inhibit the inflammatory response.

"This is one of the first demonstrations of how the microbiome can interact with epigenetic factors in the skin and modulate the skin's behavior through the inflammatory response," said George Sen, PhD, associate professor of dermatology and cellular and molecular medicine at UC San Diego School of Medicine. "Whatever environment we're facing can change a person's specific response to it. Since this epigenetic change is reversible, unlike alterations to our DNA, we can potentially control our skin inflammatory response through targeting of these enzymes."

The research was initially conducted in mouse models in which HDAC8 and HDAC9 had been genetically knocked out. As a result, the mice's skin could not tolerate microbial or viral exposures, resulting in a heightened immune reaction. The team then reproduced the findings with human cells in a culture dish.

Gallo said the work could change how doctors treat certain types of skin inflammation or other dermatologic conditions.

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To design truly compostable plastic, scientists take cues from nature

Despite our efforts to sort and recycle, less than 9% of plastic gets recycled in the U.S., and most ends up in landfill or the environment.

Biodegradable plastic bags and containers could help, but if they're not properly sorted, they can contaminate otherwise recyclable #1 and #2 plastics. What's worse, most biodegradable plastics take months to break down, and when they finally do, they form microplastics -- tiny bits of plastic that can end up in oceans and animals' bodies -- including our own.

Now, as reported in the journal Nature, scientists at the Department of Energy's Lawrence Berkeley National Laboratory (Berkeley Lab) and UC Berkeley have designed an enzyme-activated compostable plastic that could diminish microplastics pollution, and holds great promise for plastics upcycling. The material can be broken down to its building blocks -- small individual molecules called monomers -- and then reformed into a new compostable plastic product.

"In the wild, enzymes are what nature uses to break things down -- and even when we die, enzymes cause our bodies to decompose naturally. So for this study, we asked ourselves, 'How can enzymes biodegrade plastic so it's part of nature?" said senior author Ting Xu , who holds titles of faculty senior scientist in Berkeley Lab's Materials Sciences Division, and professor of chemistry and materials science and engineering at UC Berkeley.

At Berkeley Lab, Xu -- who for nearly 15 years has dedicated her career to the development of functional polymer materials inspired by nature -- is leading an interdisciplinary team of scientists and engineers from universities and national labs around the country to tackle the mounting problem of plastic landfill posed by both single-use and so-called biodegradable plastics.

Most biodegradable plastics in use today are usually made of polylactic acid (PLA), a vegetable-based plastic material blended with cornstarch. There is also polycaprolactone (PCL), a biodegradable polyester that is widely used for biomedical applications such as tissue engineering.

But the problem with conventional biodegradable plastic is that they're indistinguishable from single-use plastics such as plastic film -- so a good chunk of these materials ends up in landfills. And even if a biodegradable plastic container gets deposited at an organic waste facility, it can't break down as fast as the lunch salad it once contained, so it ends up contaminating organic waste, said co-author Corinne Scown, a staff scientist and deputy director for the Research, Energy Analysis & Environmental Impacts Division in Berkeley Lab's Energy Technologies Area.

Another problem with biodegradable plastics is that they aren't as strong as regular plastic -- that's why you can't carry heavy items in a standard green compost bag. The tradeoff is that biodegradable plastics can break down over time -- but still, Xu said, they only break down into microplastics, which are still plastic, just a lot smaller.

So Xu and her team decided to take a different approach -- by "nanoconfining" enzymes into plastics.

Putting enzymes to work

Because enzymes are part of living systems, the trick would be carving out a safe place in the plastic for enzymes to lie dormant until they're called to action.

In a series of experiments, Xu and co-authors embedded trace amounts of the commercial enzymes Burkholderia cepacian lipase (BC-lipase) and proteinase K within the PLA and PCL plastic materials. The scientists also added an enzyme protectant called four-monomer random heteropolymer, or RHP, to help disperse the enzymes a few nanometers (billionths of a meter) apart.

In a stunning result, the scientists discovered that ordinary household tap water or standard soil composts converted the enzyme-embedded plastic material into its small-molecule building blocks called monomers, and eliminated microplastics in just a few days or weeks.

They also learned that BC-lipase is something of a finicky "eater." Before a lipase can convert a polymer chain into monomers, it must first catch the end of a polymer chain. By controlling when the lipase finds the chain end, it is possible to ensure the materials don't degrade until being triggered by hot water or compost soil, Xu explained.

In addition, they found that this strategy only works when BC-lipase is nanodispersed -- in this case, just 0.02 percent by weight in the PCL block -- rather than randomly tossed in and blended.

"Nanodispersion puts each enzyme molecule to work -- nothing goes to waste," Xu said.

And that matters when factoring in costs. Industrial enzymes can cost around $10 per kilogram, but this new approach would only add a few cents to the production cost of a kilogram of resin because the amount of enzymes required is so low -- and the material has a shelf life of more than 7 months, Scown added.

The proof is in the compost

X-ray scattering studies performed at Berkeley Lab's Advanced Light Source characterized the nanodispersion of enzymes in the PCL and PLA plastic materials.

Interfacial-tension experiments conducted by co-author Tom Russell revealed in real time how the size and shape of droplets changed as the plastic material decomposed into distinct molecules. The lab results also differentiated between enzyme and RHP molecules.

"The interfacial test gives you information about how the degradation is proceeding," he said. "But the proof is in the composting -- Ting and her team successfully recovered plastic monomers from biodegradable plastic simply by using RHPs, water, and compost soil."

Russell is a visiting faculty scientist and professor of polymer science and engineering from the University of Massachusetts who leads the Adaptive Interfacial Assemblies Towards Structuring Liquids program in Berkeley Lab's Materials Sciences Division.

Developing a very affordable and easily compostable plastic film could incentivize produce manufacturers to package fresh fruits and vegetables with compostable plastic instead of single-use plastic wrap -- and as a result, save organic waste facilities the extra expense of investing in expensive plastic-depackaging machines when they want to accept food waste for anaerobic digestion or composting, Scown said.

Since their approach could potentially work well with both hard, rigid plastics and soft, flexible plastics, Xu would like to broaden their study to polyolefins, a ubiquitous family of plastics commonly used to manufacture toys and electronic parts.

The team's truly compostable plastic could be on the shelves soon. They recently filed a patent application through UC Berkeley's patent office. And co-author Aaron Hall, who was a Ph.D. student in materials science and engineering at UC Berkeley at the time of the study, founded UC Berkeley startup Intropic Materials to further develop the new technology. He was recently selected to participate in Cyclotron Road, an entrepreneurial fellowship program in partnership with Activate.

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Unlocking the mystery behind skeletal aging

 Researchers from the UCLA School of Dentistry have identified the role a critical enzyme plays in skeletal aging and bone loss, putting them one step closer to understanding the complex biological mechanisms that lead to osteoporosis, the bone disease that afflicts some 200 million people worldwide.

The findings from their study in mice, published online in the journal Cell Stem Cell, could hold an important key to developing more effective treatments for osteoporosis and improving the lives of an aging population, they say.

Cells in the bone marrow known as mesenchymal stem cells serve as the building blocks of the body's skeletal tissues, but whether these stem cells ultimately develop into bone or fat tissues is controlled in part by what are known as epigenetic factors -- molecules that regulate genes, silencing some and activating others.

The UCLA researchers, led by distinguished professor Dr. Cun-Yu Wang, chair of oral biology at the dentistry school, demonstrated that when the epigenetic factor KDM4B is absent from mesenchymal stem cells, these cells are far more likely to differentiate into fat cells than bone cells, resulting in an unhealthy imbalance that exacerbates skeletal aging and leads to brittle bones and fractures over time.

"We know that bone loss comes with age, but the mechanisms behind extreme cases such as osteoporosis have, up until recently, been very vague," said Dr Wang, the study's corresponding author and the Dr. No-Hee Park Professor of Dentistry at UCLA. "In this study, we built on more than seven years of research managed by my postdoctoral scholar and lead author Dr. Peng Deng in the hope that we can eventually prevent skeletal aging and osteoporosis."

While scientists have long understood the cellular pathway involved in bone tissue formation, the role of epigenetic factors has been murkier. Previous research by Wang, Deng and others had identified that the enzyme KDM4B plays an important epigenetic role in bone formation, but they were unsure of how its absence might affect the processes of bone formation and bone loss.

To test this, the research team created a mouse model in which KDM4B was absent or removed in several different scenarios. They found that the removal of the enzyme pushed mesenchymal stem cells to create more fat instead of bone tissue, leading to bone loss over time, which mimics skeletal aging.

In one important scenario, the scientists examined stem cell senescence, or deterioration and exhaustion -- the natural process by which mesenchymal stem cells stop rejuvenating or creating more of themselves over time. The team unexpectedly found that senescence, which leads to natural skeletal aging, was characterized by a loss of KDM4B.

In addition to age, other environmental factors are thought to reduce bone quality and exacerbate bone loss, including a high-fat diet. The team demonstrated that a loss of KDM4B significantly promoted bone loss and the accumulation of marrow fat in mice placed on a high-fat diet.

Finally, the team showed that parathyroid hormone, an anabolic drug approved by the U.S. Food and Drug Administration for the treatment of aging-related bone loss, helps to maintain the pool of mesenchymal stem cells in aging mice in a KDM4B-dependent manner.

The results not only confirm the critical role KDM4B plays in mesenchymal stem cell fate decision, skeletal aging and osteoporosis, but they show that the loss of KDM4B exacerbates bone loss under a number of conditions and, surprisingly, that KDM4B controls the ability of mesenchymal stem cells to self-renew. This study is the first in vivo research to demonstrate that the loss of an epigenetic factor promotes adult stem cell deterioration and exhaustion in skeletal aging.

The findings, the researchers say, hold promise for the eventual development of strategies to reverse bone-fat imbalance, as well as for new prevention and treatment methods that address skeletal aging and osteoporosis by rejuvenating adult stem cells.

"The work of Dr. Wang, his lab members and collaborators provides new molecular insight into the changes associated with skeletal aging," said Dr. Paul Krebsbach, dean of the UCLA School of Dentistry. "These findings are an important step towards what may lead to more effective treatment for the millions of people who suffer from bone loss and osteoporosis."

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Breaking the rules of chemistry unlocks new reaction

 Scientists have broken the rules of enzyme engineering to unlock a new method for creating chemical reactions that could unlock a wide range of new applications -- from creating new drugs to food production.

In their paper published today in Nature Catalysis, Professor Francesca Paradisi and Dr. Martina Contente of the University of Nottingham and the University of Bern show a new method to produce chemical molecules more efficiently through a new one step reaction in the enzyme.

Professor Paradis is Professor of Biocatalysis in the School of Chemistry in Nottingham and Professor of Pharmaceutical Chemistry at the University of Bern, she explains: "We have demonstrated how a very simple mutation in one of the key residues of a useful enzyme has dramatically expanded its synthetic scope, enabling the use of the mutant variant in the preparation of challenging chemical molecules, as well as natural metabolites that are vital in many biological processes in the body."

Any textbook on enzymes will report on how the catalytic amino acids in any given enzyme family are highly conserved, they are in fact a signature of the type of chemistry an enzyme can do. Variations do occur and in some cases, if the replacing amino acid is similar, both can be found in significant proportion in Nature, but others can be much less common and are found only in a limited number of species.

"In this study we have explored an untouched area of enzyme engineering and modified the a key catalytic residue in the active site of an enzyme" adds Professor Paradisi, "Previously it was thought that doing this would cause a loss of activity of the enzyme but we have found this is not the case when this biocatalyst is used in a synthetic direction and in fact challenging but very useful molecules can now be made under mild conditions which could be easily scaled up and replicated commercially for use in a wide range of products."

To change the substrate scope of an enzyme the approach has generally been to mutate the residues involved in substrate recognition, whether through rational design or directed evolution, leaving always untouched the catalytic ones.

The mutant variant of an acyl transferase enzyme was rapidly created and while the native biocatalyst would work with alcohols and linear amines, the mutant work with thiols and much more complex amines too. The research demonstrated that indeed the new variant has lost the ability to hydrolyse esters, but for synthetic applications, where an ester or other functional groups need to be made (thioesters and amides) and not cleaved, this is in fact a major advantage.

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Plastic-eating enzyme 'cocktail' heralds new hope for plastic waste

 

Plastic bottles and other waste

The scientists who re-engineered the plastic-eating enzyme PETase have now created an enzyme 'cocktail' which can digest plastic up to six times faster.

A second enzyme, found in the same rubbish dwelling bacterium that lives on a diet of plastic bottles, has been combined with PETase to speed up the breakdown of plastic.

PETase breaks down polyethylene terephthalate (PET) back into its building blocks, creating an opportunity to recycle plastic infinitely and reduce plastic pollution and the greenhouse gases driving climate change.

PET is the most common thermoplastic, used to make single-use drinks bottles, clothing and carpets and it takes hundreds of years to break down in the environment, but PETase can shorten this time to days.

The initial discovery set up the prospect of a revolution in plastic recycling, creating a potential low-energy solution to tackle plastic waste. The team engineered the natural PETase enzyme in the laboratory to be around 20 percent faster at breaking down PET.

Now, the same trans-Atlantic team have combined PETase and its 'partner', a second enzyme called MHETase, to generate much bigger improvements: simply mixing PETase with MHETase doubled the speed of PET breakdown, and engineering a connection between the two enzymes to create a 'super-enzyme', increased this activity by a further three times.

The study is published in the journal Proceedings of the National Academy of Sciences.

The team was co-led by the scientists who engineered PETase, Professor John McGeehan, Director of the Centre for Enzyme Innovation (CEI) at the University of Portsmouth, and Dr Gregg Beckham, Senior Research Fellow at the National Renewable Energy Laboratory (NREL) in the US.

Professor McGeehan said: "Gregg and I were chatting about how PETase attacks the surface of the plastics and MHETase chops things up further, so it seemed natural to see if we could use them together, mimicking what happens in nature.

"Our first experiments showed that they did indeed work better together, so we decided to try to physically link them, like two Pac-men joined by a piece of string.

"It took a great deal of work on both sides of the Atlantic, but it was worth the effort -- we were delighted to see that our new chimeric enzyme is up to three times faster than the naturally evolved separate enzymes, opening new avenues for further improvements."

The original PETase enzyme discovery heralded the first hope that a solution to the global plastic pollution problem might be within grasp, though PETase alone is not yet fast enough to make the process commercially viable to handle the tons of discarded PET bottles littering the planet.

Combining it with a second enzyme, and finding together they work even faster, means another leap forward has been taken towards finding a solution to plastic waste.

PETase and the new combined MHETase-PETase both work by digesting PET plastic, returning it to its original building blocks. This allows for plastics to be made and reused endlessly, reducing our reliance on fossil resources such as oil and gas.

Professor McGeehan used the Diamond Light Source, in Oxfordshire, a synchrotron that uses intense beams of X-rays 10 billion times brighter than the Sun to act as a microscope powerful enough to see individual atoms. This allowed the team to solve the 3D structure of the MHETase enzyme, giving them the molecular blueprints to begin engineering a faster enzyme system.

The new research combined structural, computational, biochemical and bioinformatics approaches to reveal molecular insights into its structure and how it functions. The study was a huge team effort involving scientists at all levels of their careers.

One of the most junior authors, Rosie Graham, a joint Portsmouth CEI-NREL PhD student said: "My favourite part of research is how the ideas start, whether it's over coffee, on a train commute or when passing in the university corridors it can really be at any moment.

"It's a really great opportunity to learn and grow as part of this UK-USA collaboration and even more so to contribute another piece of the story on using enzymes to tackle some of our most polluting plastics."

Read more at Science Daily

Oldest enzyme in cellular respiration isolated

 In the first billion years, there was no oxygen on Earth. Life developed in an anoxic environment. Early bacteria probably obtained their energy by breaking down various substances by means of fermentation. However, there also seems to have been a kind of "oxygen-free respiration." This was suggested by studies on primordial microbes that are still found in anoxic habitats today.

"We already saw ten years ago that there are genes in these microbes that perhaps encode for a primordial respiration enzyme. Since then, we -- as well as other groups worldwide -- have attempted to prove the existence of this respiratory enzyme and to isolate it. For a long time unsuccessfully because the complex was too fragile and fell apart at each attempt to isolate it from the membrane. We found the fragments, but were unable to piece them together again," explains Professor Volker Müller from the Department of Molecular Microbiology and Bioenergetics at Goethe University.

Through hard work and perseverance, his doctoral researchers Martin Kuhns and Dragan Trifunovic then achieved a breakthrough in two successive doctoral theses. "In our desperation, we at some point took a heat-loving bacterium, Thermotoga maritima, which grows at temperatures between 60 and 90°C," explains Dragan Trifunovic, who will shortly complete his doctorate. "Thermotoga also contains Rnf genes, and we hoped that the Rnf enzyme in this bacterium would be a bit more stable. Over the years, we then managed to develop a method for isolating the entire Rnf enzyme from the membrane of these bacteria."

As the researchers report in their current paper, the enzyme complex functions a bit like a pumped-storage power plant that pumps water into a lake higher up and produces electricity via a turbine from the water flowing back down again.

Only in the bacterial cell the Rnf enzyme (biochemical name = ferredoxin:NAD-oxidoreductase) transports sodium ions out of the cell's interior via the cell membrane to the outside and in so doing produces an electric field. This electric field is used to drive a cellular "turbine" (ATP synthase): It allows the sodium ions to flow back along the electric field into the cell's interior and in so doing it obtains energy in the form of the cellular energy currency ATP.

The biochemical proof and the bioenergetic characterization of this primordial Rnf enzyme explains how first forms of life produced the central energy currency ATP. The Rnf enzyme evidently functions so well that it is still contained in many bacteria and some archaea today, in some pathogenic bacteria as well where the role of the Rnf enzyme is still entirely unclear.

 Read more at Science Daily