De novo design of catalytic proteins

Abstract

The de novo design of catalytic proteins provides a stringent test of our understanding of enzyme function, while simultaneously laying the groundwork for the design of novel catalysts. Here we describe the design of an O(2)-dependent phenol oxidase whose structure, sequence, and activity are designed from first principles. The protein catalyzes the two-electron oxidation of 4-aminophenol (k(cat)/K(M) = 1,500 M(-1).min(-1)) to the corresponding quinone monoimine by using a diiron cofactor. The catalytic efficiency is sensitive to changes of the size of a methyl group in the protein, illustrating the specificity of the design.

Scheme 1.

Fig. 1.

Models of DF_tet.(Upper Left) Subunit composition of DF_tet A₂B₂ and DF_tet A_aA_bB₂.(Upper Right) Model of 4AP bound to DFtet A₂B₂. The carbons of the phenol are in cyan. (Lower) The solvent-accessible surface associated with DF_tet and G₄-DF_tet.

Fig. 2.

Oxidation of Fe(II) in the presence of G₄-DF_tet (circles) and L₂G₂ DF_tet (triangles) (0.15 M Mops, pH 7.0/0.15 M NaCl; 25 μM tetramer; 50 μM iron).

Fig. 3.

Binding of 4CP to diferric G₄-DF_tet (0.15 M Mops, pH 7.0/0.15 M NaCl, 25 μM tetramer).

Fig. 4.

Catalytic activity of G₄-DF_tet and variants. (A) Oxidation of 4AP catalyzed by variants of DF_tet A_aA_bB₂ and DF_tet (5 μM tetramer). The last four bars in the histogram are for DF_tet-A₂B₂ (in bold). (B) Catalysis of the oxidation of 4AP by G₄-DF_tet (squares, 100 μM) and L₂G₂-DF_tet (diamonds, 100 μM) and the background reaction (triangles). (C) Rate of oxidation of 4AP vs. substrate concentration for G₄-DF_tet (5 μM tetramer). (D) Competitive inhibition of the oxidation of 4AP by 4CP (5 μM tetramer; 0.5 mM). All reactions included 0.15 M Mops (pH 7.0), 0.15 M NaCl, 0.5 mM 4AP, 10 mM MPD, and reactions in A, C, and D included 0.5 mM 4AP.

Fig. 5.

Titration of G₄-DF_tet-A into DF_tet-B measured by the mean residue ellipticity of the solution at 222 nm. The molar ratio of the two peptides was varied, whereas the total concentration was kept constant at 5.0 μM (100 mM NaCl/25 mM Mes, pH 6.5/1 mM EDTA). The lines shown are based on a linear least square fit of the data from 0 to 0.5 and from 0.5 to 1.0 molar ratio.