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Chemists engineer metalloproteins with novel activities

New protein engineering method yields proteins capable of catalyzing reactions not seen before in biology

by Stu Borman
June 16, 2016 | A version of this story appeared in Volume 94, Issue 25

CORRECTION: This story was updated on June 17, 2016, to correct the types of reactions that the metalloproteins catalyzed.

A new technique allows researchers to create artificial metalloproteins that can catalyze reactions distinct from those of any natural protein.

Figure shows replacement of the native metal center of a metalloprotein such as myoglobin with a nonnative metal porphyrin.
Credit: Hanna M. Key
Replacing the iron center of myoglobin (left) with an iridium porphyrin and subjecting the modified protein to directed evolution produces proteins with novel activities.

The technique could “expand the toolbox of biocatalytic strategies available to chemists for asymmetric synthesis of high-value compounds such as fine chemicals and pharmaceuticals,” says Rudi Fasan of the University of Rochester, who was not involved in the work.

Chemists have previously reported methods for producing new metalloproteins, and the new strategy combines key advantages of two of those techniques.

Frances H. Arnold of Caltech and others pioneered the use of directed evolution, an iterative protein modification technique, to revise key amino acids in a metalloprotein to tweak its chemistry. But this method does not include changing the protein’s metal center to attain novel types of activity.

Another method allows for such metal center changes. It starts with a protein that lacks a metal center and then creates one by inserting a metal-cofactor complex into the binding or active site. But introducing these metal centers changes the protein’s ability to bind substrates in unexpected ways and thus makes it difficult for researchers to identify key binding-site amino acids to modify. Directed evolution has therefore not worked well with this method.

John F. Hartwig and coworkers at the University of California, Berkeley, have now melded these two techniques by replacing a metalloprotein’s native metal with a nonnative one (Nature 2016, DOI: 10.1038/nature17968). This perturbs the binding site much less than when a new metal is inserted into a non-metalloprotein. As a result, the team can more readily use directed evolution methods to further change the protein’s chemistry. Scientists have replaced metals in metalloproteins before, but in those cases the teams generally didn’t seek or obtain significantly new forms of reactivity.

In the new study, Hartwig’s group expressed in bacteria a form of myoglobin lacking its iron-heme unit. They reconstituted it with various metal porphyrins and found iridium to be the most active metal center for the type of reactivity they were seeking. They then used directed evolution to produce many versions of the iridium-substituted metalloprotein.

Some of the iridium proteins catalyze reactions that add carbenes to alkenes and α-olefins, and others convert C–H bonds into C–C bonds by enantioselective carbene insertion—the first time any proteins have catalyzed these types of reactions.

Arnold says the new work offers the exciting ability to combine protein engineering and directed evolution to create proteins with new chemistries and the exquisite selectivity enzymes offer. “A world of possibilities for nonnatural chemistry is now open,” she says.



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