Biomedicine

Enzymes Built from Scratch

Researchers engineer never-before-seen catalysts using a new computational technique.

In a major step forward for computational protein design, scientists have built from scratch a handful of enzymes that successfully catalyze a specific chemical reaction. These proteins have no naturally occurring counterparts, and the reaction–which breaks down a man-made chemical–has no natural catalyst.

Catalyst creativity: Researchers developed a computational technique to build enzymes from scratch. An enzyme called retro-aldolase, a portion of which is shown above, was designed to break carbon-carbon bonds in a non-natural chemical substrate (yellow and white stick model). The gray mesh is the enzyme’s active site, its geometry carefully crafted to hold the substrate in place. The orange and green stick models indicate components of the enzyme that are particularly important in prodding the reaction forward.

“It makes it clear that we can compute a structure that will catalyze a reaction where there was none before,” says Frances Arnold, professor of chemical engineering and biochemistry at Caltech, who was not involved in the research. Arnold calls new enzymes the “holy grail” of computational protein design. Designing any protein from scratch is a tall order; engineering a protein that can carry out a given function requires far more sophistication.

David Baker and his colleagues at the University of Washington focused on a reaction that would break certain bonds between carbon atoms. The ability to design enzymes that can break and make carbon-carbon bonds could potentially enable scientists to break down environmental toxins, manufacture drugs, and create new fuels.

As they report in the journal Science, Baker and his group first designed what an ideal active site would look like for the reaction. An active site is a pocket within an enzyme where the catalyzed reaction takes place. In order to do its job, an active site must have precise geometry and chemical makeup, tailored to the reaction it catalyzes. Some components hold the reacting molecules in place, while others participate in the reaction’s chemical mechanisms.

Once the researchers computed the active site, they used a newly developed set of algorithms to model proteins that have such a site. Each designed protein was ranked according to its ability to bind the reacting chemicals and hold them in the proper position.

The next step was to actually synthesize the selected proteins. The researchers derived gene sequences for 72 of the designed enzymes, ordered snippets of DNA containing those genes, and used bacteria to turn the genes into proteins. Each protein was then tested for its ability to catalyze the carbon-carbon bond breaking reaction.

Of the 72 proteins selected, 32 successfully helped along the reaction. The most efficient proteins sped up the reaction to 10,000 times the rate without an enzyme.

While that’s an impressive feat compared with earlier enzyme design attempts, the synthesized enzymes pale in comparison to naturally occurring ones. “It’s not very good at all,” says Baker. “Naturally occurring enzymes can increase the rate of reactions by much, much greater amounts”–as much as a quadrillion-fold.

“One of our research problems is to figure out what’s missing from our designs that naturally occurring enzymes have figured out,” says Baker. In follow-up studies, his group has taken two approaches to this problem: refining its computer algorithms, and asking nature to step in where the researchers left off. By using their minimally functional enzymes as evolutionary starting points, the researchers can use directed evolution to create more efficient catalysts.

In the past, directed evolution has been an alternative approach to creating desirable enzymes. But Baker believes that it can be used as a complement to computational approaches. Computational design gives researchers a way to build proteins from the ground up, allowing them to engineer enzymes for reactions that have no natural counterpart. “That way, we would be free from the tyranny of having to find something in nature to start from,” says Arnold, whose work has focused heavily on directed evolution.

But directed evolution provides a means of making those structural tweaks that the computational designs algorithms aren’t yet sophisticated enough to handle. “It’s actually the wave of the future,” says Baker, “because these directed-evolution experiments can capture much more subtle things than we can capture in the calculations.”

Baker’s is not the first group to tackle computational enzyme design. For example, Caltech biochemist Steve Mayo, a pioneer in computational protein design, reported the creation of enzymes from existing nonenzyme proteins in 2001. But Baker’s approach differs in that it doesn’t use existing proteins as starting points–it’s true de novo design.

Arnold says that Baker’s enzymes are also more powerful than Mayo’s, but that it’s hard to nail down precisely how much more. “It’s a different kind of enzyme, so you can’t really compare apples and oranges,” she says. “But this is a pretty good apple.”

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