Giant Enzymes in Tiny Organisms Synthesize Giant Toxins

Tilting the scales at 4.7 megadaltons and reaching 1 micron in length, the newly discovered algal enzyme PKZILLA-1 is the largest protein ever identified in biology. Indeed, PKZILLA-1 is 25% larger than the previous record holder, a muscle protein called titin. PKZILLA-1 even has a big sidekick, PKZILLA-2, which weighs in at 3.2 megadaltons. Whereas PKZILLA-1 contains 140 enzyme domains, PKZILLA-2 contains 99.

But why are the PKZILLAs so big? And why does the bigness of PKZILLAs matter?

Apparently, the PKZILLAs became supersized through evolution so that they could synthesize supersized polyketide polyether toxins called prymnesins. These toxins, which are among the largest nonpolymeric compounds in nature, come from Prymnesium parvum algae, which cause massive environmental fish kills.

The bigness of PKZILLAs matters because it could help marine biologists improve their monitoring of harmful agal blooms. For example, monitoring efforts could focus on the genes for the prymnesins rather than the toxins themselves. And contemplating PKZILLAs’ bigness could encourage us to think bigger biological thoughts.

“This is the Mount Everest of proteins,” said Bradley Moore, a marine chemist with joint appointments at Scripps Oceanography and Skaggs School of Pharmacy and Pharmaceutical Sciences. “This expands our sense of what biology is capable of.” Moore is the senior author of a new study describing how the PKZILLAs were discovered. This study, titled, “Giant polyketide synthase enzymes in the biosynthesis of giant marine polyether toxins,” appeared recently in the journal Science.

“[The] biosynthesis of these massive microalgal toxins has remained an enigma despite a wealth of intimate knowledge of polyketide biochemistry from decades of research in bacteria and fungi,” Moore and his colleagues wrote. To unravel the enigma, they applied a customized gene annotation strategy that enabled the discovery of two massive polyketide synthase genes, PKZILLA-1 and -2, from P. parvum strain 12B1 that we propose are responsible for the complete backbone assembly of its notorious ladder-frame polyether toxin, prymnesin-1.

“The discovery of the PKZILLAs and their role in prymnesin biosynthesis lays the foundation for the development and implementation of alternative linked omics approaches to fully uncover the complete suite of prymnesin biosynthetic enzymes. Moreover, PKZILLAs offer the opportunity to dissect the enzymology of ladder-frame polyether biosynthesis and will serve as a model to capture and dissect giant genes, transcripts, and proteins in specialized metabolism.”

“Understanding how nature has evolved its chemical wizardry gives us as scientific practitioners the ability to apply those insights to creating useful products, whether it’s a new anticancer drug or a new fabric,” Moore emphasized.

P. parvum commonly known as golden algae, is an aquatic single-celled organism found all over the world in both fresh and saltwater. Blooms of golden algae are associated with fish die-offs due to its toxin prymnesin, which damages the gills of fish and other water breathing animals. In 2022, a golden algae bloom killed 500–1,000 tons of fish in the Oder River adjoining Poland and Germany. The microorganism can cause havoc in aquaculture systems in places ranging from Texas to Scandinavia.

Prymnesin belongs to a group of toxins called polyketide polyethers that includes brevetoxin B, a major red tide toxin that regularly impacts Florida, and ciguatoxin, which contaminates reef fish across the South Pacific and Caribbean. These toxins are among the largest and most intricate chemicals in all of biology, and researchers have struggled for decades to figure out exactly how microorganisms produce such large, complex molecules.

Beginning in 2019, Moore and colleagues began trying to figure out how golden algae make their toxin prymnesin on a biochemical and genetic level. The scientists began by sequencing the golden alga’s genome and looking for the genes involved in producing prymnesin. Traditional methods of searching the genome didn’t yield results, so the team pivoted to alternate methods of genetic sleuthing that were more adept at finding super long genes.

“We were able to locate the genes, and it turned out that to make giant toxic molecules this alga uses giant genes,” said Vikram Shende, a postdoctoral researcher in Moore’s lab at Scripps and co-first author of the paper.

With the PKZILLA-1 and PKZILLA-2 genes located, the team needed to investigate what the genes made to tie them to the production of the toxin. Timothy Fallon, a postdoctoral researcher in Moore’s lab at Scripps and co-first author of the paper. said the team was able to read the genes’ coding regions like sheet music and translate them into the sequence of amino acids that formed the protein.

When the researchers completed this assembly of the PKZILLA proteins they were astonished at their size. After additional tests showed that golden algae actually produce these giant proteins in life, the team sought to find out if the proteins were involved in making the toxin prymnesin. The PKZILLA proteins are technically enzymes, meaning they kick off chemical reactions, and the team played out the lengthy sequence of 239 chemical reactions entailed by the two enzymes with pens and notepads.

“The end result matched perfectly with the structure of prymnesin,” Shende said.

Following the cascade of reactions that golden algae uses to make its toxin revealed previously unknown strategies for making chemicals in nature, Moore said. “The hope is that we can use this knowledge of how nature makes these complex chemicals to open up new chemical possibilities in the lab for the medicines and materials of tomorrow,” he added.

Finding the genes behind the prymnesin toxin could allow for more cost-effective monitoring for golden algae blooms. Such monitoring could use tests to detect the PKZILLA genes in the environment akin to the PCR tests that became familiar during the COVID-19 pandemic. Improved monitoring could boost preparedness and allow for more detailed study of the conditions that make blooms more likely to occur.

Fallon said the PKZILLA genes the team discovered are the first genes ever causally linked to the production of any marine toxin in the polyether group that prymnesin is part of.

Next, the researchers hope to apply the non-standard screening techniques they used to find the PKZILLA genes to other species that produce polyether toxins. If they can find the genes behind other polyether toxins, such as ciguatoxin which may affect up to 500,000 people annually, it would open up the same genetic monitoring possibilities for a suite of other toxic algal blooms with significant global impacts.