Artificial Enzyme Can Catalyze Reactions

A dawning field of research, artificial biology, is working toward creating a genuinely new organism. At Princeton, chemistry professor Michael Hecht and the researchers in his lab are designing and building proteins that can fold and mimic the chemical processes that sustain life. Their artificial proteins, encoded by synthetic genes, are approximately 100 amino acids long, using an endlessly varying arrangement of 20 amino acids.

Now, Hecht and his colleagues have confirmed that at least one of their new proteins can catalyze biological reactions, meaning that a protein designed entirely from scratch functions in cells as a genuine enzyme.

Once Hecht and his research team had successfully created artificial proteins for E. coli, they began looking for critical functions that they could disrupt in these simple bacteria. They found four genes that, when removed, would not only render the E. coli inert -- effectively dead -- but which their artificial proteins could then "rescue," or resuscitate.

They first identified these artificial proteins in 2011, and they have spent the past six years working to figure out the precise mechanisms by which their new proteins functioned, now detailed in a Jan. 15 paper in Nature Chemical Biology.

It's important not to assume that an artificial protein will work the same way as the natural one whose deletion it is rescuing, Hecht cautioned.

Determining the mechanisms their artificial proteins used took countless experiments. "We had four different gene deletions -- four different enzymatic functions," said Ann Donnelly, lead author on the paper.

After years of experiments, the team had concluded that two of these "rescues" operate by replacing enzymes -- proteins that serve to catalyze other reactions, helping them operate quickly enough to sustain life -- with proteins that were not enzymes themselves, but which boost the production of other processes in the cell, she said. The third was showing progress, but the fourth had frustrated multiple researchers who came through Hecht's lab.

But then Donnelly, who was a graduate student when she did the research and is now a research specialist in bioinformatics at the University of Pittsburgh, cracked the code, realizing that the artificial protein, Syn-F4, was actually an enzyme. Out of the original set of proteins that could rescue gene deletions, this is the only one that has turned out to be an enzyme -- at least so far, she said.

"We have a completely novel protein that's capable of sustaining life by actually being an enzyme -- and that's just crazy," Hecht said.

Hecht's team had created a strain of E. coli that was missing the enzyme Fes, without which it cannot access the iron needed to sustain life. "We all need iron," Hecht said. "Even though iron is abundant on earth, biologically accessible iron is not." Cells have developed molecules like enterobactin, he explained, which can scavenge iron from any available source, but they then need a tool -- like Fes -- to wrest the iron from the tight grip of the enterobactin.

This modified E. coli strain had no way to extract, or hydrolyze, the iron from its enterobactin, until it was "rescued" by Syn-F4. The researchers had provided iron to the E. coli, but it only stained the cells red, since although they could accumulate the bound metal, they could not liberate it from enterobactin or access it for cellular use.

"And then Ann noticed ... they aren't red anymore, they're white, which suggests the cells can break this down and get the iron, which suggests we actually have an enzyme!" said Hecht.

Researchers are on the cusp of a true synthetic biology, Hecht said.

"E. coli has 4,000 different genes," he said. "We didn't test all 4,000, because the only way this experiment works is if nothing grows on minimal medium, and of the 4,000, that's only true for some.

"We're starting to code for an artificial genome. We've rescued 0.1 percent of the E. coli genome. ... For now, it's a weird E. coli with some artificial genes that allow it to grow. Suppose you replace 10 percent or 20 percent. Then it's not just a weird E. coli with some artificial genes, then you have to say it's a novel organism."

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