Mighty enzymes

molecule
Summer 2017
Queen’s University’s David Zechel studies microbial­ enzymes­ that can break down the controversial herbicide­ glyphosate, also known as Roundup.

David Zechel of Queen’s University

David Zechel of Queen’s University. 

Is it possible for chemists to make the perfect molecule? David Zechel, associate professor at Queen’s University Department of Chemistry in Kingston, Ont., says there is one — glyphosate. “It is probably as close as chemistry can come to creating a silver bullet in terms of a molecule that is very specific in its effects,” says Zechel, who researches natural product biosynthesis and enzyme catalysis.

Glyphosate is also a perfect storm in terms of what it symbolizes. First brought to market as a key ingredient in the common herbicide Roundup, manufactured by Monsanto, today glyphosate is present in products sold by many chemical companies, including Syngenta and Dow AgroSciences. It is one of the most widely applied pesticides in the world: in 2012 approximately 122,000 to 132,000 tonnes were applied in the US alone, according to the United States Environmental Protection Agency, which also states it has been the most-used active pesticide ingredient since 2001.

In the public sphere, glyphosate is deeply polarizing. While the agriculture sector relies upon glyphosate to help feed an ever-growing global human population, environmental groups have raised concerns about its potential links to cancer, particularly certain types of lymphoma.  Reports that glyphosate residue has been found in human breast milk, bread and beer have sown distrust and even animosity towards the global chemical giants producing the compound. Some nations within the European Union, as well as a network of non-governmental organizations, have been pushing for its ban. In early 2015 a report from the World Health Organization’s International Agency for Research on Cancer stated that glyphosate was “probably carcinogenic to humans,” sparking further petitions opposing its use. Later that same year, the European Food Safety Authority (EFSA) reported that glyphosate was “unlikely to pose a carcinogenic hazard to humans,” prompting further debate and responses from groups on both sides of the issue. 

The debate is set to heat up once again this summer, with the European Commission, the independent executive arm of the EU that draws up proposals for new European legislation, expected to issue a proposal on the future of glyphosate. The compound’s ultimate fate in the European Union will be decided by year’s end. But given the amount of attention the compound has drawn, it’s likely the fight will continue in other jurisdictions for years to come.

RoundUp

Zechel believes there is a middle ground between meeting the needs of the agriculture industry and protecting public safety. Through phosphonate biochemistry research, Zechel is striving to find ways to mimic the enzymes in bacteria that naturally break down glyphosate, a phosphonic acid. Understanding the glyphosate-degrading pathway could allow for enhanced bioremediation of glyphosate-contaminated areas, possibly ameliorating the compound’s unsavoury reputation. “Degrading these herbicides more aggressively in the soil before they have the chance to wash into the water supply — I think that would be helpful,” Zechel says. 

When it comes to the question about glyphosate and human health, Zechel tends to side with the non-toxic camp. One argument he finds compelling has to do with the route of exposure: glyphosate is highly water soluble and therefore unlikely to accumulate in fatty tissue. This also means that, in contrast to other pesticides such as DDT, glyphosate does not bioaccumulate as it moves up the food chain. Toxicity, however, “is a relative term, so what is not toxic to us at a certain age can be toxic to a fetus or another organism,” Zechel says.

Researchers from around the globe, including Canada, have been investigating the possible effects of glyphosate-based herbicides in various ecosystems. For example, Leanne F. Baker of the University of New Brunswick and her team have investigated the effects of glyphosate on chironomids (lake flies) in wetlands. In a 2014 article in Environmental Toxicology and Chemistry, they found that while the herbicide by itself had no direct effect on the emergence of lake flies, it could affect aquatic plant cover, which led to indirect effects on fly populations. Another study by Baker’s team, published in 2016 in Ecotoxicology, found that the herbicide could also affect the plankton community but only when applied in combination with fertilizers. Moreover, these effects become evident long after application, when glyphosate residues were no longer detectable in surface water.
Glyphosate is effective because of what it does — as well as doesn’t — do. In plants, it targets the enzyme 5-enoylpyruvylshikimate 3-phosphate (EPSP) synthase, which is critical to synthesizing the amino acids phenylalanine, tyrosine and tryptophan. Without these amino acids, which serve as chemical building blocks for enzymes and other proteins, the plant dies. 

But glyphosate really came into its own once Monsanto scientists were able to engineer genetically modified crops that were immune to the effects of glyphosate. These crops, dubbed “Roundup Ready,” included soy, corn, canola, alfalfa and cotton and were introduced in 1996. Farmers could now spray glyphosate over the entire field, knowing that only the desired plants would survive. This sparked an exponential increase in the use of glyphosate worldwide. 

As with many polarized issues, the reality about glyphosate is more complex than the ongoing debate might suggest. Zechel says that within most soils there are abundant microorganisms that can break glyphosate down within a few days to several weeks to degrade glyphosate. That said, problems can arise when glyphosate gets washed into rivers and lakes and becomes broadly distributed in the water supply. Here, it takes longer to degrade because the bacteria that break them down are less common in water than in soil.
Such microbes, says Zechel, “ are starved for phosphate.” Often referred to as the staff of life, phosphate is critical to the chemical backbone of both DNA and RNA, not to mention the energy-carrying molecule adenosine triphosphate and the phospholipids that make up all cell membranes. So it’s not surprising that various organisms have evolved mechanisms for turning phosphonic acids — including glyphosate — into inorganic phosphate. The trick for scientists, therefore, is to harness this ability found in nature to reduce the potential impact of glyphosate in areas where it might have accumulated. Such a strategy would be preferable to an outright glyphosate ban, says Zechel, which could result in the creation of “something far worse taking its place, simply because it’s so hard to improve on its mechanism of action, particularly in combination with genetically modified plants.” 

Zechel describes the two-stage decomposition of glyphosate. First, microorganisms in the soil split the glyphosate into two products. One is glyoxylate, a simple organic compound that is further degraded by other organisms in the soil. The other product is aminomethylphosphonic acid (AMPA), which is similar enough to glyphosate to raise some of the same safety concerns. AMPA contains the same carbon-phosphorous bond that was at the heart of the original glyphosate molecule. Because the bond is stable and hard to break, AMPA can stick around in the soil long after the herbicide itself has disappeared. 

However, organisms exist that are capable of breaking the C-P bond found in both glyphosate and AMPA. By breaking this bond using carbon-phosphorus lyase (CP-lyase), they release the inorganic phosphate that is critical to life. Zechel has long been fascinated with what he calls the “really crazy enzyme reactions” that enable soil microbes to pull off this trick. “It’s amazing that these organisms come up with cool chemistry to cleave this carbon-phosphorous bond — a neat expression of evolution,” he says. 

Zechel explains that CP-lyase pathway involves multiple enzymes, which posed a significant challenge for the team. A breakthrough involved collaborating with University of Copenhagen microbiologist Bjarne Hove-Jensen, whose work helped establish that one of the enzymes in the pathway, PhnP, performed a key step in recycling the product of the CP-bond cleavage. 

Zechel’s research has brought to light other remarkable findings. He has recently characterized a new, two-enzyme pathway that cleaves C-P bonds. His team has expressed these genes, phnY and phnZ, purified the encoded enzymes and replicated the same reaction in vitro. Unexpectedly, they found that oxygen was involved in the CP-bond cleaving step performed by phnZ. “The reaction was happening through oxidation,” says Zechel. “Nevertheless, it was still following chemical principles. What I really like about this is that nature doesn’t necessarily have to reinvent the wheel when coming up with new enzyme chemistry. It can take an existing enzyme structure and just tweak it a little bit.” 

To Zechel, this example of chemical innovation illustrates the boundless potential of what he calls “dark matter,” which refers to the more than 99 percent of microbes that do not easily grow in a laboratory and are thus relatively unknown to science. “The environmental extremes that microbes are subjected to have led to the evolution of breathtaking chemistry. This enables catabolism of molecules into nutrients, or the synthesis of complex molecules for defence, such as antibiotics.” This vast pool of new enzyme reactions and novel molecules could “replenish our current arsenal of antibiotics, which is becoming less and less effective due to overuse and increasing pathogen resistance.” 

Because the microbes don’t survive long enough outside their natural habitat to be studied, Zechel and his team study them using metagenomic DNA. The process involves taking a soil sample and extracting all the genetic material from everything living in it, such as worms and bacteria. This technique has been made possible by the plummeting cost of genome sequencing; Zechel says that new microbial genome sequences are uploaded daily to the GenBank, a sequence database provided by the National Center for Biotechnology Information. Comparing new sequences with known organisms in this database allows researchers like Zechel to find genes encoding novel enzymes used for a wide variety of catabolic or anabolic processes. “We can get right in there and express that gene, isolate that enzyme and try to get it to work in the lab,” he says.

So what does the future hold for these enzymes? “Highly proficient glyphosate-degrading enzymes could be introduced into a soil bacterium that could be used to inoculate contaminated soils,” says Zechel. “One could also consider using isolated enzymes that are immobilized on filters that could be used to process contaminated water.”

That said, Zechel believes that the amelioration of environmental problems shouldn’t rely solely upon the possibility of finding more cool enzymes. Basic green chemistry principles prescribe more prudent use of herbicides, including glyphosate. For example, Zechel agrees with municipal bylaws that limit the use of glyphosate on golf courses, private lawns or other places where aesthetics are the main goal. Agriculture is a different story but even there, Zechel believes that only the minimum amount required should be used. “These companies want to sell more of this stuff but they should be showing an awareness of social responsibility,” Zechel says. “I would like to see this in chemical companies where they are aware their products can be damaging. They should get on top of it and show concrete evidence that they are trying to mitigate it.”  

Glyphosate and the monarch butterfly

Apart from the human health concerns, another potential impact of widespread glyphosate use is the loss of certain wild plants that are perceived as weeds in agriculture. One example is milkweed, the only plant that monarch caterpillars feed upon. In a 2012 article in Insect Conservation and Diversity, Iowa State University biologist John M. Pleasants and University of Minnesota biologist Karen S. Oberhauser reported that monarch populations, which winter in Mexico and migrate north into the United States and southern Canada in the warmer months, have been declining since about the mid-1990s due largely to a reduction in milkweed. They estimate that the monarch population is at about 20 percent of its numbers before the mid-1990s. However, glyphosate is likely only one of many factors involved. A 2016 article by Cornell University­ biologists published in Oikos pointed to sparse autumnal nectar sources, climate change and habitat fragmentation as possible stressors of monarch butterflies.