Hecht Team Elucidates Structure of Novel Enzyme that “Works”
Shuffling protein sequences around to create novel arrangements, a team of scientists led by the Hecht Lab discovered a de novo enzyme that supports the very life-sustaining activity of its biological doppelgänger.
It was the first time a synthetic, or lab-created, enzyme had ever been found to do this. Researchers named their enzyme Syn-F4, for synthetic ferric enterobactin esterase.
The research has its roots in a 2018 paper published in Nature Chemical Biology announcing the finding. But while significant back then, the enzyme’s structure was still shrouded in mystery. It precipitated a four-year investigation that has finally borne fruit.
This week, in collaboration with structural biologists at Shinshu University in Japan and a former postdoc under Hecht, the team lays out the fully articulated structure, Crystal structure and activity of a de novo enzyme, ferric enterobactin esterase Syn-F4, in the Proceedings of the National Academy of Sciences (PNAS).
Professor of Chemistry Michael Hecht (center) with paper authors Brendan Sperling (left) and Guanyu Liao, both graduate students in the Hecht Lab.
“It’s alternative to life as we know it,” said Professor of Chemistry Michael Hecht of the de novo enzyme. “It came from no ancestry. It’s made from scratch, and we finally have an alternative 3D structure to show it.”
The structure confirms that Syn-F4 acts as a catalyst to liberate iron from siderophores, iron-chelating compounds. All living creatures need iron to survive. Normally, cellular e coli acquires iron through an enzyme, Fes, whose task is to release iron from siderophores, thereby making it accessible to an organism. Without Fes, the iron cannot be accessed and the organism would die.
The Hecht Lab’s de novo enzyme serves the same purpose of liberation as Fes. In other words, said Hecht, “It works.”
In collaboration with Ryoichi Arai, the former postdoc, the crystal structure of Syn-F4 was found to form a dimeric 4-helix bundle with a hole in the central region surrounded by residues essential for enzymatic activity in vivo. This suggests an active site within the structure, information that was not known at the time of the 2018 paper.
“The structure of Syn-F4 shows that its structure and biochemical mechanism differ substantially from those of natural enterobactin esterases,” said Arai, now an associate professor at Shinshu University. “It thereby demonstrates that proteins which did not arise in living systems can perform life-sustaining enzymatic functions in ways dramatically different from those that evolved from common ancestry in nature.”
Lab’s Mission: Find Novel Proteins
The Hecht Lab designs de novo protein sequences from “scratch” to find alternatives to the way nature has evolved enzymes to function.
Their investigation deleted a gene in an e coli sample that codes for a particular protein, in this case, the enzyme that releases iron from siderophores. Placing cells—minus the native gene—in a medium, researchers waited to see what would happen. If any of the de novo genes could rescue the deleted function, it would enable the growth of a colony.
But Hecht is not interested simply in novel sequences that give rise to a colony. He wants novel sequences that actually do something a native enzyme does. And in Syn-F4, he found one.
This graphic shows the overall crystal structure of novel Syn-F4. Note the tunnel at the center of the structure on the bottom illustration, which researchers say is surrounded by residues essential for enzymatic activity.
“My lab is interested in making these sequences that are unrelated to anything that’s ever been on earth. They’re novel,” said Hecht. “But if it’s just making sequences, who cares? The question is, can they do anything? Or to ask the even more profound question, can they sustain life? Can they provide a function without which the organism is dead?
“If there are 4,000 genes in e coli, one could do this experiment 4,000 times. It can fail 3,999 times and work once and one can still get a Ph.D. We’ve done a lot of looking,” Hecht added. “Ann [Donnelly] showed that Syn-F4 is a bona fide catalyst in her paper back in 2018, but how does it work? Well, we didn’t know that until we could see the structure atom by atom through Ryoichi’s work.”
The structure shows the putative active site and enzymatic mechanism of Syn-F4.
“The mechanism that Syn-F4 hydrolyzes ferric-enterobactin is this,” said Guanyu Liao, an author on the paper and graduate student in the Hecht Lab. “Histidine and glutamate in the active site form a catalytic dyad, which hydrogen-bonds and therefore activates a water molecule to attack the ester bond in the substrate.
“The novelty of the mechanism is that the enzyme does not use serine to attack the ester bond in ferric-enterobactin, in contrast to the mechanism of natural enterobactin-hydrolyzing enzymes.”
This research was supported by funding from the National Science Foundation to Michael Hecht (MCB-1947720).