Molecular mechanism of strict substrate specificity of an extradiol dioxygenase, DesB, derived from Sphingobium sp. SYK-6

Abstract

DesB, which is derived from Sphingobium sp. SYK-6, is a type II extradiol dioxygenase that catalyzes a ring opening reaction of gallate. While typical extradiol dioxygenases show broad substrate specificity, DesB has strict substrate specificity for gallate. The substrate specificity of DesB seems to be required for the efficient growth of S. sp. SYK-6 using lignin-derived aromatic compounds. Since direct coordination of hydroxyl groups of the substrate to the non-heme iron in the active site is a critical step for the catalytic reaction of the extradiol dioxygenases, the mechanism of the substrate recognition and coordination of DesB was analyzed by biochemical and crystallographic methods. Our study demonstrated that the direct coordination between the non-heme iron and hydroxyl groups of the substrate requires a large shift of the Fe (II) ion in the active site. Mutational analysis revealed that His124 and His192 in the active site are essential to the catalytic reaction of DesB. His124, which interacts with OH (4) of the bound gallate, seems to contribute to proper positioning of the substrate in the active site. His192, which is located close to OH (3) of the gallate, is likely to serve as the catalytic base. Glu377' interacts with OH (5) of the gallate and seems to play a critical role in the substrate specificity. Our biochemical and structural study showed the substrate recognition and catalytic mechanisms of DesB.

Figure 1. Catalytic reaction of DesB.

(A) The chemical structure of gallate and the catalytic reaction of DesB. (B) The chemical structures of PCA and 3MGA.

Figure 2. Structure of DesB.

(A) The crystal structure of the DesB dimer. The asymmetric unit of the crystal contains the whole dimer. Subunit A is shown in rainbow colours along the polypeptide chain from the N (blue) to the C (red) terminus. Subunit B is shown in light grey. (B) The Fe (II) coordination sphere of DesB and an anomalous difference Fourier map (contour level of 5.0 σ). (C) The active site of the anaerobic DesB-gallate complex. Carbon atoms of bound gallate are shown in orange. Hydrogen bonds are indicated by pink dotted lines with distances in Å units. (D) Comparison of the coordination spheres between DesB (carbon atoms in green) and LigAB (carbon atoms in yellow) in the substrate (PCA) complex forms . Carbon atoms of gallate are shown in orange.

Figure 3. Active site structures of the DesB-gallate and DesB-PCA complexes.

(A) Crystal preparation scheme of the anaerobic DesB-gallate complex (upper panel) and alternative conformations of the Fe (II) ion observed in the anaerobic DesB-gallate complex (lower panel). An anomalous difference Fourier density (blue) is contoured at the 5.0 σ level. (B) Omit maps for A- and R-site Fe (II) ions (5 σ.. The green and red densities are omit maps for the Fe (II) ions at the A- and R-sites, respectively. (C) Crystal preparation scheme of the aerobic DesB-gallate complex (upper panel) and an anomalous difference Fourier map (5.0 σ) of the aerobic DesB-gallate complex (lower panel). Thin white sticks and small white spheres indicate the positions of gallate and the Fe ion found in the anaerobic DesB-gallate complex, respectively. (D) Crystal preparation scheme of the anaerobic DesB-PCA complex (upper panel) and its active site structure with an anomalous difference Fourier map (5.0 σ) (lower panel). Carbon atoms of PCA are shown in yellow. The thin stick model in orange indicates the position of gallate in the anaerobic DesB-gallate complex.

Figure 4. His124 forms a hydrogen bond with a hydroxyl group of gallate and PCA.

(A) Superposition of the active sites of DesB in the substrate-free (white), anaerobic PCA-complex (carbon atoms in yellow) and anaerobic gallate-complex (carbon atoms in green) forms. Carbon atoms of gallate are shown in orange. (B) Crystal preparation scheme of the anaerobic His124Phe DesB-gallate complex (upper panel) and its active site structure with an anomalous difference Fourier map (blue, 5.0 σ) (lower panel). A simulated annealing omit map for gallate (cyan) is also shown (3.0 σ). The thin stick model in pink indicates the position of gallate in the anaerobic DesB-gallate complex.

Figure 5. Substrate recognition and Fe (II) shift in the catalytic reaction of DesB.

The light green circle represents the productive binding site for gallate. The pink circle represents the productive coordination sphere with the Fe (II) ion at the A-site.