Tag: enzymes

Understanding bacteria’s role in transforming steroids to pharmaceuticals

Understanding bacteria’s role in transforming steroids to pharmaceuticals

Identifying 3D structure of enzymes by University of Guelph researchers key first step in harnessing alterations for disease treatments.

For decades, pharmaceutical companies have been using bacteria found in soil and water to chemically convert steroids into effective treatments for human diseases. One example is cortisol, which is used to treat asthma and skin rashes. But how bacteria convert steroids is not fully understood.

Now a research team from the University of Guelph has taken a significant step forward in answering that question. Using the Canadian Light Source (CLS) at the University of Saskatchewan, Dr. Stephen Seah and colleagues have determined the 3D structures of steroid-transforming enzymes from Proteobacteria (also called Pseudomonadota), a large, diverse family of gram-negative bacteria named after Proteus – the shape-shifting Greek sea god.Video: Understanding bacteria’s role in transforming steroids to pharmaceuticals

Studying the 3D structure of these enzymes, which Seah says would be impossible without the ultrabright X-ray source of the CLS, is key to understanding how this Proteobacteria chemically transforms steroids – such as bile acids – which are typically resistant to being changed.

Seah and his colleagues found that the bacteria have evolved to transform steroids as a means to obtain carbon and energy for their own growth. However, he says, these transformations can be harnessed to chemically alter steroids into compounds that we can use for disease treatments; a discovery that will help advance future pharmaceutical development.

“If we understand the process, we can manipulate other bacteria to produce novel compounds that may have medicinal properties,” says Seah. “I think my work helps fill in this gap of knowledge.” The team’s research findings were published recently in both the Journal of Biological Chemistry and Biochemistry.

This new research, says Seah, also opens the door to exploring the potential of other enzymes in bacteria to change the chemical structure of steroids. “In other words, one could create steroids with diverse chemical structures using the many steroid-modifying enzymes that bacteria produced to alter naturally occurring steroids,” he says. “Some of these modified steroids may have therapeutic properties.”

Read more on the CLS website

Image: Protein structure

Credit: CLS

How Enzymes Make New Products Using Greenhouse Gases

How Enzymes Make New Products Using Greenhouse Gases

Humans have been using enzymes to create new products for thousands of years. First it was wine, then cheese. In this tradition, three years ago, a team of scientists tweaked a lyase (HACL/S) to reverse course. Instead of breaking, the enzyme synthesizes novel chemicals through the addition of carbon atoms. 

Now, using the Advanced Photon Source (APS), a U.S. Department of Energy (DOE) Office of Science user facility at DOE’s Argonne National Laboratory, an international team shows how HACL/S enzymes work on an atomic level. Their findings can serve as the basis for increasing the enzymes’ yield and versatility while drawing down as precursors atmospheric carbon dioxide and methane.

HACL/S enzymes were originally discovered for their role in breaking down fatty acids into formyl-CoA (fCoA) and an aldehyde or ketone in mammalian peroxisomes. Since then, scientists have discovered their ability to condense fCoA with various aldehydes and ketones and have one carbon atom added to them. Given the enzyme’s ability to reverse reaction direction from a lyase to a synthase, combined with an abundance of carbon molecules in the atmosphere, HACL/S is an ideal model for biocatalytic production of a variety of new products.  

However, compared to chemical synthetic reactions, biocatalytic production usually produces low yield. The authors of the current research reasoned that if they could manipulate the specificity of these enzymes to accept different kinds of ketones or aldehydes, they could boost the enzymes’ productivity and efficiency.

In order to do that, they first needed to discover how these enzymes worked.

To begin, the team chose from the list of over 100 newly identified proteins six variants of the enzyme that exhibited high activity with aldehyde compounds of different length and formyl-CoA and had amino acid sequences that were diverse enough to cover the HACL/S subfamily. The team synthesized genes for each of the variants, then expressed them in Escherichia coli bacteria.

After purifying the expressed proteins, some members of the international team characterized the enzymes biochemically. Others produced crystals of five enzymes separately and in complexes with acyl-CoA substrates, ThDP cofactor, and ADP. They X-rayed the crystals, diffracted to 1.70–2.70 Å, at beamlines 19-ID – the Structural Biology Center (SBC) – and 23-ID-B – the National Institute of General Medical Sciences and National Cancer Institute Structural Biology Facility (GM/CA) – of the Advanced Photon Source (APS) at Argonne National Laboratory. 

The crystal structures obtained from the X-ray data revealed what computer-predicted models could not: a flexible loop on the C terminus that locked on the cofactor and kept it bound to the enzyme’s active site. When the substrate was added, the loop closed the active site, stabilizing the cofactor and enabling the transfer of the formate compound to the substrate. 

Read more on Argonne website

Gut enzymes may explain differential disease and FDA-approved drug outcomes

Gut enzymes may explain differential disease and FDA-approved drug outcomes

Our bodies need neurotransmitters and hormones to stay healthy, but too much or too little can cause conditions such as breast cancer or Parkinson’s disease. Normally, excess neurotransmitters and hormones in the body are removed through excretion via the gut. A team of scientists has discovered a new class of enzymes from bacteria in our guts that can alter levels of serotonin, the “feel good” neurotransmitter, and estradiol, a sex hormone, among other compounds. The scientists also found that certain FDA-approved drugs can inhibit these bacterial enzymes. In this way, a cancer drug may inadvertently cause depression in some people by interfering with excretion and thereby initiating a change in their serotonin levels.

These surprising findings could explain why some people respond well to certain drugs and other people don’t, leading the way to more personalized drug dosing based on genomic analysis of the patient and the microbes in their gut. The researchers used the Advanced Photon Source (APS), a U.S. Department of Energy (DOE) Office of Science user facility at DOE’s Argonne National Laboratory.

Our bodies maintain equilibrium in part by ensuring that detrimental substances, such as environmental toxins or excess molecules created naturally when we eat too much turkey at Thanksgiving, are flushed away. To do this, the liver attaches a sugar to the unwanted molecule that serves as a “tag” for trafficking it to the gut for excretion.

For the past 10-15 years, many scientists have focused their investigations on one detrimental substance in particular—drugs that cause adverse reactions in the GI tract—to discover what makes them toxic. They found that certain microbes living in the gut feed off the sugar attached to the detrimental substance by using an enzyme that removes the sugar for microbial growth. Rather than being excreted, the detrimental substance, freed of its sugar – or “reactivated,” in scientific language – remained in the body, causing off-target effects, from irritable bowel syndrome to Crohn’s disease.

Little was known, however, about how gut microbes were behaving toward naturally-occurring molecules like hormones or neurotransmitters. To fill that gap, the research team turned their attention to dopamine and serotonin, as well as estradiol and thyroid hormones, to see if the gut microbes were processing them the way they processed toxic drugs.

A primary question was: Why do the bacteria have these enzymes in the first place? 

Through structural biology, in vitro biochemistry, multi-omics, and in vivo studies, the team showed that specific enzymes in the gut acted on these naturally occurring molecules in the same way they processed man-made molecules like drugs. This suggested to the scientists that sugar-linked natural chemicals like hormones and neurotransmitters play an important role in the microbial evolution of an enzyme that allows gut bacteria to take advantage of this resident food supply.

The enzyme in question is called GUS, or beta-glucuronidase. Previous research had shown that certain types of FDA-approved drugs, including those that fight cancer and depression, inhibit a specific subset of gut microbial GUS enzymes. Different people have different types of microbes in their guts and, therefore, different GUS enzymes. The scientists wondered whether this could explain why different people react differently to these drugs: Might the difference lie in which enzymes were being inhibited and which enzymes were left to interfere with the body’s natural chemical balance, or homeostasis?  

The key answers lay in detailed studies using structural biology, a field that investigates how complex biological macromolecules do their job. Drugs usually have one target, but in the expansive gut microbiome, hundreds of different proteins can all do the same job. The scientists set out to understand on an atomic level why some GUS are more active than others.

Using the National Institute of General Medical Sciences and National Cancer Institute Structural Biology Facility (GM/CA) beamlines at 23-ID-B and 23-ID-D at the APS, the team collected data that enabled them to solve the crystal structures of various species of gut microbes in complex with various anticancer and antidepressant drugs. What they found not only surprised them but also doubled the pool of enzymes that matter – they’d discovered that a whole other class of enzymes, called C-Terminal Domain GUS (CTD), are critically efficient at processing the sugar-attached molecules and are very potently inhibited by certain drugs.

Read more on Argonne website

Image: Simpson et al. pinpoint the gut microbial enzymes (green) that reactivate neurotransmitters and hormones (yellow, orange, and purple) essential to homeostasis and to diseases ranging from cancer to anxiety. They also show that a range of FDA-approved drugs (blue) inhibit these enzymes and impact local and systemic hormone and neurotransmitters levels. The study highlights the indispensable role of gut microbes in endobiotic homeostasis and indicates that therapeutic disruption of this role promotes interindividual variabilities in drug response.

Taking candid shots of radical proteins

Taking candid shots of radical proteins

Scientists capture how radical electrons influence protein structure before they have time to react

Some enzymes in the body carry radicals, chemical groups with highly reactive unpaired electrons, to catalyse biochemical reactions, but it has proven challenging to study the structure of these enzymes with the radicals intact. The X-ray crystallography techniques conventionally used to study protein structure introduce ‘X-ray damage’ that would neutralise radicals and alter the protein structure. To study how radicals influence proteins, researchers turned to a ribonucleotide reductase enzyme subunit called R2 that uses a radical to synthesise DNA bases. The team previously used X-ray crystallography at beamline I24 and small angle X-ray scattering at beamline B21 of the Diamond Light Source to solve the structure of this enzyme without safeguarding the radical. In the recent study, they harnessed X-ray free electron laser (XFEL) serial femtosecond crystallography at the Linac Coherent Light Source in collaboration with Diamond’s XFEL Hub to zero in on the radical. With XFEL, they used X-rays to rapidly capture the structure of the protein within femtoseconds — 1015 times quicker than a second and too quick for X-rays to neutralise the radical or distort protein structure. By comparing the enzyme with and without the radical, they revealed that the presence of the unpaired electron greatly influences the structure of the enzyme’s active site. Their research will allow them to explore the workings of this and related enzymes in finer detail and holds promise for designing drugs that target radical enzymes in cancer cells and infectious microbes.

Read more on the Diamond website

Image credit: Martin Högbom

Enzymes that Convert Plant Fiber into Simple Sugars

Enzymes that Convert Plant Fiber into Simple Sugars

Research from Lawrence Berkeley National Laboratory (Berkeley Lab), Lawrence Livermore National Laboratory (LLNL),  and UC Davis sheds new light on how to access the sugars locked up in plants to produce petroleum-free fuels, chemicals, and medicines.

Using microbes to convert grasses, weeds, wood, and other plant residues into sustainable products will be key to achieving carbon neutrality and could even help eliminate drug shortages. But cellulose, the tough tissue that makes up a large proportion of herbaceous and woody plant bodies, is hard to break down into its composite sugars, which the microbes need to build other molecules. Only organisms that have evolved specialized enzymes, or those that host microbiomes of those organisms, are able to get sugars from cellulose-rich plant matter.

Scientists are studying how these enzymes work so that they can develop more efficient methods to convert plant waste into sweet ingredients.

“We want to utilize plant residues and there is a lot of plant waste out there,” said project co-leader Tina Jeoh, a professor of biological and agricultural engineering at UC Davis. “These sugars are key to establishing a bioeconomy built on cycling renewable carbon for biofuel, biochemical, and biomaterial alternatives to fossil fuel-sourced versions.”

Jeoh and her colleagues used a technique developed at the Berkeley Synchrotron Infrared Structural Biology (BSISB) Imaging Program. The technique combines a novel microfluidic device and infrared spectroscopy to study how a cellulose-degrading enzyme works in real time. Their work was recently published in the journal Green Chemistry.

Cellulose is composed of many glucose molecules, each joined together by a single covalent bond. The long chains of glucose are twisted together into complex rope-like structures called fibrils, which stay in that formation thanks to many hydrogen bonds between the tightly-organized glucoses. Scientists have hypothesized that these hydrogen bonds are the reason why cellulose-chopping enzymes are so slow – they act as obstacles blocking access to the covalent bond.

To finally understand exactly what is happening during these reactions, a pair of Berkeley Lab researchers designed an experimental system that could provide information about how the atomic structure of the cellulose changed while the enzyme was working. The system consists of a small disk-like device that holds a tiny quantity of fluid containing cellulose, from green algae, and tiny amount of an enzyme derived from a fungi. The device moves the two fluids together, allowing the reaction to begin, in the path of a powerful beam of infrared light generated by the Berkeley Lab’s Advanced Light Source (ALS). Detectors near the device then measure how the light was absorbed by the combined fluids at different time intervals when exposed to the beam. Changes in spectral features will indicate changes in the chemical bonds or bond environments in the molecules.

The results from this method, a type of operando spectroscopy methodology that uses Fourier transform infrared spectromicroscopy, indicate that the hydrogen bonds in the fibrils are indeed acting as roadblocks for the enzymes.

Read more on Berkeley Lab website

Image : A microscopy image of plant cells showing their rigid walls, which are given structure by cellulose.

Credit : Rattiya Thongdumhyu/Shutterstock

Blood-type conversion process informed by crystallography now in pre-clinical trials

Blood-type conversion process informed by crystallography now in pre-clinical trials

Application of a discovery that was aided in part by the Canadian Light Source (CLS) at the University of Saskatchewan has advanced to pre-clinical trials and is now the basis of a dynamic new startup.

In 2019 Dr. Stephen Withers and colleagues at the University of British Columbia identified a series of enzymes that can be used to modify the chemical structure of a sugar antigen on the surface of blood cells, thereby converting a Type A blood cell to a Type O blood cell — the universal donor type. The team used crystallography on the CMCF beamline at the CLS to better understand how the enzymes cause this change.

These same antigens are also present on the surface of solid organs, and Withers and colleagues have demonstrated that the enzymes they discovered are very efficient at making this conversion both on the surface of red blood cells and on the surface of donated human organs such as lungs or kidneys.

Avivo – the company launched to bring this technology to the marketplace – is now busy finetuning both applications. If successful, this exciting advance would be a huge step forward in addressing shortages in blood and organ supply here in Canada and around the world. “The idea is that we could broaden the supply considerably,” says Withers, a professor in the Departments of Chemistry and Biochemistry and the Michael Smith Laboratories at UBC. “It would remove the need to worry about blood type in transfusions (and organ donations).”

John Barclay, VP of business development with Avivo says the company is focusing first on applying their approach to organ donations because it’s considerably more straightforward to remove the conversion enzymes prior to transplantation than it is to remove them before transfusing blood.

When a donor organ is harvested, it will often be placed on a perfusion device that continuously pumps a preservation solution, or perfusate, through it to maintain the tissue’s viability. The enzymes discovered by the Withers team can be added to the fluid mixture, where they essentially convert the blood type of the organ to the universal blood type. After that conversion, the solution – including the enzymes — is essentially “rinsed” out of the organ as part of the existing transplant process. Removing the enzymes from red blood cells or whole blood is considerably more challenging, says Barclay.

The Avivo team has demonstrated the process works using a set of human lungs that were deemed not viable for transplanting into a patient. “We’ve shown that we can remove those antigens and convert an A type lung to an O type lung quite readily,” says Withers. “We’re working on kidneys at the moment…so that’s very exciting.”

This application of their technique is in pre-clinical trials now; they’re hoping to move on to clinical trials (i.e., in human patients) in 2024.

How the Canadian Light Source contributed

“The information we learned from it (crystallography) was very supportive in knowing exactly the structure of the enzymes we’re adding,” says Withers. This information, he says, will be very useful if they need to modify the structure of the enzyme.

It will also be valuable when they seek regulatory approval, to be able to present the complete structure of the enzymes. “We’ve learned a lot more through having that information, which may be useful in the future,” says Withers.

Read more on the CLS website

Image: Steve Withers, John Barclay, and John Coleman.