The Bare Bones of Catalysis

Researchers have used test-tube evolution to create a new, smaller enzyme that still performs the function of its natural counterpart. This strategy of stripping an enzyme or other protein to its bare essentials, described in today's issue of Science, could reveal the way particular kinks and folds dictate how a protein works. The technique might also lead to tiny molecules that retain a therapeutic protein's function while lasting longer in the body.

Biochemists have been harnessing an artificial version of evolution to refine natural enzymes, making new variants that work faster, longer, and at higher temperatures. Now a team led by chemist Donald Hilvert of the Swiss Federal Institute of Technology in Zürich, working at The Scripps Research Institute in La Jolla, California, has taken this approach in a slightly different direction by trimming down an enzyme. The researchers' target was chorismate mutase (CM), an enzyme that helps bacteria and higher plants make certain amino acids. CM is a dimer: two identical monomers, each consisting of three helices, locked in a tight embrace. Hilvert's group set out to part them and force a single monomer to develop the dimer's ability to catalyze a chemical reaction needed to make the amino acids.

Splitting the dimer required the researchers to bend one of the helices into a "U" by inserting a new turn. Because it is nearly impossible to predict how a turn will alter the structure or function of a helix, Hilvert's team members decided to let natural selection pick a winner. They created a "library" of DNA sequences encoding millions of versions of the enzyme, each one with a different turn, and slipped these genes into a strain of Escherichia coli that can't make CM. Next, they added a selection pressure: The bacteria were grown on food lacking the amino acids that CM helps to make. This meant that bacteria with monomers that work like the dimer would flourish, while ill-equipped bacteria would perish. "We let the organisms fight it out," says Hilvert. They found that 0.05% of the variants were monomers that could work as well as the original dimeric CM.

"It's an impressive accomplishment," says Frances Arnold, a chemical engineer at the California Institute of Technology in Pasadena who has applied evolution to protein design. Recreating an enzyme's function in a molecule with a different structure, Arnold adds, "is a very difficult design problem--and he let nature tell him what the answers are." This kind of redesign may also have practical payoffs--among them, getting valuable proteins to stop clumping in industrial vats.

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