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Design and Discovery: Enzyme Reprogramming

SBKB [doi:10.1038/sbkb.2012.124]
Featured Article - February 2013
Short description: Rational structure-based design of a de novo Due Ferri protein alters its substrate and reactivity.

Surface models of DFsc and G4DFsc. The active site was made more accessible in the latter by mutation of four Ala to Gly (shown in white). 1

The goal of de novo protein design is to build a protein with a predicted structure and activity from scratch, exploring the important and subtle relationship between structure and function. Working on a previously de novo designed enzyme, DeGrado and colleagues (PSI NESG) have now taken a further step: by modifying its active site, they reprogrammed its substrate and catalytic activity.

The authors started with the single-chain Due Ferri protein (DFsc), a four-helix bundle designed to mimic the activity of natural dioxygen-activating di-iron enzymes that catalyze the oxidation of hydroquinones. First, four alanine residues along the active-site channel were mutated to glycine. The resulting construct, G4DFsc, had a more accessible active site and catalyzed the oxidation of hydroquinones with less formation of a nonproductive iron-bound complex.

To alter the enzyme's catalytic activity, from oxidation of hydroquinones to N-hydroxylation of arylamines, the authors made several mutations in the active site of G4DFsc, using the natural p-aminobenzoate N-oxygenase AurF as a guide. AurF contains an additional histidine residue involved in coordinating one of the iron atoms. Because mutation of the corresponding residue in DFsc to histidine would produce steric clashes with neighboring residues, the authors used a computational scanning technique to identify additional mutations that would stabilize the new histidine. They ultimately made three mutations, going out to the third shell of interaction with the histidine residue to create a hydrogen-bonding network that mimicked that in the active site of AurF. As expected, the resulting construct, 3His-G4DFsc, catalyzed the N-hydroxylation of arylamines while rates of hydroquinone oxidation were near background. The NMR structure of a more stable variant of 3His-G4DFsc (PDB 2LFD), containing two glycine residues in the active-site channel instead of four, indicated that the overall tertiary structure of the protein remained intact.

The reprogramming of G4DFsc using rational and computational methods highlights the ability to perform different reactions using the same protein scaffold and paves the way for further studies into the roles of specific amino acids in catalysis.

Jennifer Cable

References

  1. A.J. Reig et al. Alteration of the oxygen-dependent reactivity of de novo Due Ferri proteins.
    Nat. Chem. 4, 900-6 (2012). doi:10.1038/nchem.1454

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