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Volume 87 Issue 49 | p. 9 | News of The Week
Issue Date: December 7, 2009

Metalloprotein Made To Order

Bioinorganic Chemistry: Researchers construct metalloenzyme active site in another protein
Department: Science & Technology
Keywords: nitric oxide (NO), 2O)">nitrous oxide (N2O), nitric oxide reductase, myoglobin, protein design
Pocket Change
Experimentally determined crystal structure of NOR-like active site designed into a myoglobin pocket. Green sphere is Fe(II); dotted lines show close interactions among Fe, amino acids, and water (red sphere); and flat structure with green center is heme.
Credit: © 2009 NATURE
8749NOTW_p9pocket
 
Pocket Change
Experimentally determined crystal structure of NOR-like active site designed into a myoglobin pocket. Green sphere is Fe(II); dotted lines show close interactions among Fe, amino acids, and water (red sphere); and flat structure with green center is heme.
Credit: © 2009 NATURE

Researchers have used rational design to create a synthetic metalloprotein with an active site that looks and acts like that of the native version. The working model of nitric oxide reductase (NOR) was created by introducing an iron binding site into a completely different protein (Nature, DOI: 10.1038/nature08620).

NOR, which catalyzes the reduction of NO to N2O, is a key enzyme in the nitrogen cycle and is essential to life. The ability to re-create NOR activity in another protein improves scientists’ understanding of its structure and function and could have implications for studies of the greenhouse effect (N2O is a greenhouse gas) and other processes.

Although a number of rationally designed proteins have been created before, few have been both structural and functional models of native proteins. And the design of metalloproteins—the active sites of which are difficult to understand, much less mimic—has likewise been rare.

Bioinorganic chemist Yi Lu of the University of Illinois, Urbana-Champaign, and coworkers created the NOR model in myoglobin, an enzyme that has a binding pocket with the right dimensions and correct shape to accommodate the active site. They introduced into that pocket two histidines and one glutamate. Two other key components, a third histidine and a heme group, were already present there.

A crystal structure they obtained shows that the redesigned site matches the group’s computer model and is a near-exact replica of NOR’s active site. The redesigned myoglobin catalyzes NO reduction, albeit at a lower level of activity than NOR.

Lu notes that the ability to study NOR “has been slowed by the lack of a good expression system to make a large amount of active enzyme as well as the lack of a high-resolution crystal structure.” The mimic, he says, “provides an excellent model system for understanding NOR and could have practical applications in biological and environmental systems.” He adds that the work also helps prove that glutamate is essential for both iron binding and NOR activity, a proposal that has been a matter of debate.

Metalloenzyme specialist Joan S. Valentine of the University of California, Los Angeles, points out that “rational design of functional metal-free enzymes has had some outstanding successes in recent years, [but] design of functional metalloenzymes has lagged behind—mainly, I think, because investigators have been pessimistic about the possibilities of success at our current level of understanding of structure-function relationships in metalloenzymes.”

This new paper “will have considerable impact because it demonstrates that the rational design approach can successfully produce metalloenzymes that mimic not only the structures of natural metalloenzymes but also their catalytic reactivities,” she says. “I believe it will inspire other workers to begin programs to design other new metalloenzymes and thus expand the field.”

 
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