The muscle protein titin has long been the titan of the molecular world. Built of more than 34,000 amino acids and weighing in at a hefty 3.7 megadaltons, titin has long been considered the largest protein in the world—until now.
A new group of toxin-making enzymes has been discovered inside the algae Prymnesium parvum, one of which—christened PKZILLA-1—dwarfs titin by more than 10,000 amino acids. P. parvum uses the monstrous PKZILLAs to manufacture a toxin that it secretes into the water, posing a threat to fish and human health alike.
Biochemist Timothy Fallon, Ph.D., is mainly interested in giant toxic molecules produced by single-celled aquatic organisms called dinoflagellates, which are a common cause of harmful algal blooms known as red tides. But dinoflagellates have massive genomes, which makes them difficult to study. Working with Moore, Fallon turned to P. parvum as a simpler study system because it has a much smaller genome but produces a similar toxin, called prymnesin.
Prymnesin is a polyketide, built from a chain of ketone and methylene groups. Fallon scoured the genome of P. parvum for polyketide synthesis (PKS) genes to try to find the builder of prymnesin, but automated processes for identifying genes within genomes can be error-prone.
To be sure he didn’t miss anything, Fallon manually ran potential PKS gene segments through databases to confirm their identity and hit a stroke of good luck.
Bits and pieces of the PKZILLAs were scattered throughout the sequence data, so Fallon painstakingly assembled the genes over time. Once he had the genes, he could translate them into the proteins they code for—a puzzle he finally completed several years ago.
The next step was confirming that the enormous PKZILLA genes are actually turned into proteins in living cells. The researchers freeze-dried the algae and chopped up all their proteins into fragments using enzymes. Sequencing those fragments revealed that at least 75% of PKZILLA-1 and 76% of PKZILLA-2 are in fact turned into protein. This approach doesn’t allow them to confirm that PKZILLAs exist as single, massive proteins, but Fallon is confident that they do.
Further evidence that PKZILLAs exist as single contiguous proteins comes from their function. Biochemist Vikram Shende, Ph.D., also of UCSD, analyzed the proteins and identified the chemical reactions that different sections of it would produce. “These two giant proteins, you can almost read them like a book,” Moore said. “The coolest thing is being able to, as a chemist, read that sequence and draw a chemical structure of what each of these enzymes is doing.” The molecule that Shende predicted PKZILLA makes is strikingly similar to prymnesin, supporting the idea that the giant toxin is indeed made by these equally giant proteins.
