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How a Soil Microbe Could Rev up Artificial Photosynthesis

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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. 

Credit: H. DeMirci et al., ACS Central Science, 2022

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 Vöhringer-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.

Wakatsuki said his team continues to collaborate with Erb’s group, and it’s working with the LCLS sample delivery group and with researchers at the SLAC-Stanford cryogenic electron microscopy (cryo-EM) facilities to find a way to make this approach work.

Researchers from the RIKEN Spring-8 Center and Japan Synchrotron Radiation Research Institute also contributed to this work, which received major funding from the DOE Office of Science. Much of the preliminary work for this study was carried out by SLAC summer intern Yash Rao; interns Brandon Hayes, E. Han Dao and Manat Kaur also made key contributions. DOE’s Joint Genome Institute provided the DNA used to produce the ECR samples. SSRL, LCLS, the Advanced Photon Source and the Joint Genome Institute are all DOE Office of Science user facilities.

Citation: Hasan DeMirci et al., ACS Central Science, 25 April 2022 (10.1021/acscentsci.2c00057)

SLAC is a vibrant multiprogram laboratory that explores how the universe works at the biggest, smallest and fastest scales and invents powerful tools used by scientists around the globe. With research spanning particle physics, astrophysics and cosmology, materials, chemistry, bio- and energy sciences and scientific computing, we help solve real-world problems and advance the interests of the nation.

SLAC is operated by Stanford University for the U.S. Department of Energy’s Office of Science. The Office of Science is the single largest supporter of basic research in the physical sciences in the United States and is working to address some of the most pressing challenges of our time.

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Original Article: bioengineer.org

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Gene Linked to Severe Learning Disabilities Governs Cell Stress Response

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DURHAM, N.C. – A gene that has been associated with severe learning disabilities in humans has been found to also play a vital role in cells’ response to environmental stress, according to a Duke University study appearing May 24 in the journal Cell Reports.

DURHAM, N.C. – A gene that has been associated with severe learning disabilities in humans has been found to also play a vital role in cells’ response to environmental stress, according to a Duke University study appearing May 24 in the journal Cell Reports.

Cells are stressed by factors  that may damage them, such as extreme temperatures, toxic substances, or mechanical shocks. When this happens, they undergo a range of molecular changes called the cellular stress response.

“Every cell, no matter from which organism, is always exposed to harmful substances in their environment that they have to deal with all the time,” said Gustavo Silva, assistant professor of biology at Duke and senior author on the paper. “Many human diseases are caused by cells not being able to cope with these aggressions.”

During the stress response, cells press pause the genes related to their normal housekeeping activities, and turn on genes related to crisis mode. Just like in a house being flooded, they put down the window cleaner, turn off the TV, and run to close the windows, then they patch holes, turn on the sump pump, and if needed, rip up carpet and throw away irreparably damaged furniture.

While studying mechanisms related to the cells’ health and their response to stress, the team saw that, under stress, a group of proteins was being modified inside the cells. They dug into it and found that the master regulator of this process is a gene called Rad6.

“When there is a stressor, cells need to change what proteins are produced,” said Vanessa Simões, associate in research in the Silva lab and lead author of the paper. “Rad6 goes in and gets the (protein-building) ribosomes to change their program and adapt what they are producing for the new stressful circumstances.”

Rad6 isn’t just any random gene. It can be found, sometimes under a different name, in almost all multicellular organisms. In humans, it is known for its association with a set of symptoms called “Nascimento Syndrome,” that include severe learning disabilities.

Nascimento Syndrome, also called X-linked intellectual disability type Nascimento, is still a poorly understood disease. It was officially described in 2006, and tends to run in families, giving scientists an early clue to its genetic causes. Affected individuals have severe learning disabilities, characteristic facial traits, with wide-set eyes and a depressed nose bridge, and a range of other debilitating symptoms.

Like many other genes, Rad6 doesn’t just do one thing. It’s a multiuse tool. By discovering an additional function, and one so tightly related to the cell’s health, Silva and his team get to add a new piece to the puzzle of Nascimento Syndrome.

“It’s still a big question or how exactly can a mutation to this gene lead to such a drastic syndrome in humans,” said Silva. “Our findings are exciting because Rad6 can be a model on which we can do genetic manipulations to try to understand how problems in coping with harmful conditions can be connected to how this disease progresses.”

“If we get a better understanding of how this gene works, we can actually try to interfere with it to help these patients have a better outcome.” he said.

But how does one actually “look” at what is happening with an infinitesimally small protein when a cell is stressed? With a fair amount of teamwork. Simões and Silva paired up with researchers from the Duke Biochemistry department and the Pratt School of engineering to gather all the help they needed.  

“We used biochemistry analyses, cellular assays, proteomics, molecular modeling, cryo-electron microscopy, a whole set of advanced techniques,” said Silva.

“It’s the cool thing about being in a place like Duke,” he said. “We found collaborators and resources easily, right here, and that really increases the impact of a study and our ability to do a more complete work.”

Funding for this study was provided by US National Institutes of Health R00 Award ES025835 and R35 Award GM137954 to Gustavo Silva. This work was also supported in part by R01 Award GM141223 to Alberto Bartesaghi and the Intramural Research Program of the NIH, National Institute of Environmental Health Sciences Grant ZIC ES103326 to Mario J. Borgnia. Cryo- EM work was performed at the Duke University Shared Materials Instrumentation Facility (SMIF), a member of the North Carolina Research Triangle Nanotechnology Network (RTNN), which is supported by the National Science Foundation (grant ECCS- 1542015) as part of the National Nanotechnology Coordinated Infrastructure (NNCI). Funding was also provided from the UNC Lineberger Comprehensive Cancer Center through the University of California, Riverside Fund and the Cancer Center Support Grant P30CA016086. 

CITATION: “Redox-Sensitive E2 1 Rad6 Controls Cellular Response to Oxidative Stress Via K63-Linked Ubiquitination of Ribosomes,” Vanessa Simões, Blanche K. Cizubu, Lana Harley, Ye Zhou, Joshua Pajak, Nathan A Snyder, Jonathan Bouvette, Mario J. Borgnia, Gaurav Arya, Alberto Bartesaghi, and Gustavo M. Silva. Cell Reports, May 24 2022. DOI: 10.1016/j.celrep.2022.110860

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New Light-powered Catalysts Could Aid in Manufacturing

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CAMBRIDGE, MA — Chemical reactions that are driven by light offer a powerful tool for chemists who are designing new ways to manufacture pharmaceuticals and other useful compounds. Harnessing this light energy requires photoredox catalysts, which can absorb light and transfer the energy to a chemical reaction.

CAMBRIDGE, MA — Chemical reactions that are driven by light offer a powerful tool for chemists who are designing new ways to manufacture pharmaceuticals and other useful compounds. Harnessing this light energy requires photoredox catalysts, which can absorb light and transfer the energy to a chemical reaction.

MIT chemists have now designed a new type of photoredox catalyst that could make it easier to incorporate light-driven reactions into manufacturing processes. Unlike most existing photoredox catalysts, the new class of materials is insoluble, so it can be used over and over again. Such catalysts could be used to coat tubing and perform chemical transformations on reactants as they flow through the tube.

“Being able to recycle the catalyst is one of the biggest challenges to overcome in terms of being able to use photoredox catalysis in manufacturing. We hope that by being able to do flow chemistry with an immobilized catalyst, we can provide a new way to do photoredox catalysis on larger scales,” says Richard Liu, an MIT postdoc and the joint lead author of the new study.

The new catalysts, which can be tuned to perform many different types of reactions, could also be incorporated into other materials including textiles or particles.

Timothy Swager, the John D. MacArthur Professor of Chemistry at MIT, is the senior author of the paper, which appears today in Nature Communications. Sheng Guo, an MIT research scientist, and Shao-Xiong Lennon Luo, an MIT graduate student, are also authors of the paper.

Hybrid materials

Photoredox catalysts work by absorbing photons and then using that light energy to power a chemical reaction, analogous to how chlorophyll in plant cells absorbs energy from the sun and uses it to build sugar molecules.

Chemists have developed two main classes of photoredox catalysts, which are known as homogenous and heterogenous catalysts. Homogenous catalysts usually consist of organic dyes or light-absorbing metal complexes. These catalysts are easy to tune to perform a specific reaction, but the downside is that they dissolve in the solution where the reaction takes place. This means they can’t be easily removed and used again.

Heterogenous catalysts, on the other hand, are solid minerals or crystalline materials that form sheets or 3D structures. These materials do not dissolve, so they can be used more than once. However, these catalysts are more difficult to tune to achieve a desired reaction.

To combine the benefits of both of these types of catalysts, the researchers decided to embed the dyes that make up homogenous catalysts into a solid polymer. For this application, the researchers adapted a plastic-like polymer with tiny pores that they had previously developed for performing gas separations. In this study, the researchers demonstrated that they could incorporate about a dozen different homogenous catalysts into their new hybrid material, but they believe it could work more many more.

“These hybrid catalysts have the recyclability and durability of heterogeneous catalysts, but also the precise tunability of homogeneous catalysts,” Liu says. “You can incorporate the dye without losing its chemical activity, so, you can more or less pick from the tens of thousands of photoredox reactions that are already known and get an insoluble equivalent of the catalyst you need.”

The researchers found that incorporating the catalysts into polymers also helped them to become more efficient. One reason is that reactant molecules can be held in the polymer’s pores, ready to react. Additionally, light energy can easily travel along the polymer to find the waiting reactants.

“The new polymers bind molecules from solution and effectively preconcentrate them for reaction,” Swager says. “Also, the excited states can rapidly migrate throughout the polymer. The combined mobility of the excited state and partitioning of the reactants in the polymer make for faster and more efficient reactions than are possible in pure solution processes.”

Higher efficiency

The researchers also showed that they could tune the physical properties of the polymer backbone, including its thickness and porosity, based on what application they want to use the catalyst for.

As one example, they showed that they could make fluorinated polymers that would stick to fluorinated tubing, which is often used for continuous flow manufacturing. During this type of manufacturing, chemical reactants flow through a series of tubes while new ingredients are added, or other steps such as purification or separation are performed.

Currently, it is challenging to incorporate photoredox reactions into continuous flow processes because the catalysts are used up quickly, so they have to be continuously added to the solution. Incorporating the new MIT-designed catalysts into the tubing used for this kind of manufacturing could allow photoredox reactions to be performed during continuous flow. The tubing is clear, allowing light from an LED to reach the catalysts and activate them.

“The idea is to have the catalyst coating a tube, so you can flow your reaction through the tube while the catalyst stays put. In that way, you never get the catalyst ending up in the product, and you can also get a lot higher efficiency,” Liu says.

The catalysts could also be used to coat magnetic beads, making them easier to pull out of a solution once the reaction is finished, or to coat reaction vials or textiles. The researchers are now working on incorporating a wider variety of catalysts into their polymers, and on engineering the polymers to optimize them for different possible applications.

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The research was funded by the National Science Foundation and the KAUST Sensor Initiative.

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Watching Video Feed of Hospitalized Baby Improves Pumping Experience

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Parents who used videoconferencing technology to view their hospitalized baby reported an improved pumping experience while expressing milk for their premature infant. Videoconferencing also helped the whole family connect to their infant in the Neonatal Intensive Care Unit (NICU). These findings were published in Breastfeeding Medicinethis month.

Parents who used videoconferencing technology to view their hospitalized baby reported an improved pumping experience while expressing milk for their premature infant. Videoconferencing also helped the whole family connect to their infant in the Neonatal Intensive Care Unit (NICU). These findings were published in Breastfeeding Medicinethis month.

“Breast milk feeding is an essential component of care for the hospitalized premature infant, but it can be challenging due to factors including low milk supply, the need to express milk instead of feeding directly from the breast, as well as the stress and anxiety for new parents who are physically separated from their premature infants in the hospital environment,” said study lead author Adrienne Hoyt-Austin. “Our study explored the experience of pumping milk while watching one’s hospitalized baby with videoconferencing.”

The UC Davis Health study enrolled parents who used FamilyLink when they are not at the bedside in the UC Davis NICU. FamilyLink is a videoconferencing program which gives families the option to see their baby through a secure connection from a home computer, tablet or cellphone 24/7.

The team interviewed participants who pumped breastmilk while using FamilyLink to view their infant and those who pumped without videoconferencing.

Participants had given birth to an infant who was less than 34 weeks gestational age and was admitted to the UC Davis NICU.

In a one-on-one interview, participants were asked 14 open-ended questions regarding their breast milk pumping experience. The qualitative analysis identified four common themes. It showed that videoconferencing:

Provided bonding and connection. Participants felt “more of a connection” and “more of a bond” when seeing their hospitalized infant on video.
Provided motivation to pump. One participant said that seeing their baby is a “visual reminder that this is what I’m doing this for.”
Reminded participants that they were separated from their baby. One participant said, “I became just kind of guilty watching, feeling like I should be there instead of away.”
 Connected the whole family to their baby. Participants reported that videoconferencing helped introduce new family members to the baby and explain the complicated issue of neonatal hospitalization.

“In our interviews, we heard over and over again that that videoconferencing improved the pumping experience and gave motivation to continue to provide breast milk for their hospitalized infant. Participants also felt that seeing their baby while pumping strengthened the bond between the family with their newborn,” said Hoyt-Austin. “We hope that the use of videoconferencing for NICU parents will become a more widely available tool in NICUs that can help new parents in their breastfeeding journey.”       

The study co-authors are Iesha Miller, Kara Kuhn-Riordon, Jennifer Rosenthal, Caroline Chantry, James Marcin, Kristin Hoffman and Laura Kair, all of UC Davis Health.

The project was funded by the Children’s Miracle Network at UC Davis and the Clinical and Translational Science Center Highly-Innovative Award (UL1-TR001860). The researchers were supported by HRSA T32HP30037 grant, NIH’s Building Interdisciplinary Research Careers in Women’s Health (BIRCWH) award (K12 HD051958) and Eunice Kennedy Shriver National Institute of Child Health and Human Development (NICHD) K23HD1015-50 grant.

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