Crystal structure and mutagenic analysis of GDOsp, a gentisate 1,2‐dioxygenase from Silicibacter Pomeroyi

Abstract

Dioxygenases catalyze dioxygen incorporation into various organic compounds and play a key role in the complex degradation pathway of mono-and polycyclic aromatic and hetero-aromatic compounds. Here we report the crystal structure of gentisate 1,2-dioxygenase from Silicibacter pomeroyi (GDOsp) at a 2.8 Å resolution. The enzyme possessed a conserved three-dimensional structure of the bicupin family, forming a homotetramerization. However, each subunit of GDOsp unusually contained two ferrous centers that were located in its two homologous cupin domains, respectively. Further mutagenic analysis indicated that the enzyme activity of GDOsp depends on the microenvironment in both metal-binding sites. Moreover, homologous structural comparison and functional study on GDOsp variants unveiled a group of functionally essential residues and suggested that the active site of the enzyme is located in the amino-terminal domain, but could be influenced by changes in the carboxyl domain. Therefore, GDOsp may provide a working model for studying long-distance communication within a protein (or its complex).

Ring-Cleaving Dioxygenases with a Cupin Fold

“…Salicylate is not converted but acts as a competitive inhibitor (55,56). All GDOs characterized to date belong to the bicupin family (1,20). Each bicupin subunit of the homotetrameric enzyme from Escherichia coli contains a mononuclear iron center coordinated to three His ligands, leaving three solvent-occupied sites on the iron available for interactions with substrate (1).…”

“…Cleavage of the O-O bond and insertion of one oxygen atom into the ring, promoted by ketonization of the hydroxyl substituent at C-5, would generate a cyclic lactone that may be hydrolyzed by the hydroxide that after O 2 cleavage remained at the iron (1,20,56). The mechanism proposed for GDO is very similar to that of the type I extradiol dioxygenases (cf.…”

The salicylate 1,2‐dioxygenase as a model for a conventional gentisate 1,2‐dioxygenase: crystal structures of the G106A mutant and its adducts with gentisate and salicylate

“…The side chains of Arg127, His162 and Arg83 undergo significant displacement from their original positions, as seen in the substrate adduct structures of wild-type SDO. Accordingly, the same conformational changes caused by the movement of Arg83 towards the gentisate molecule were observed: closure of the loop corresponding to residues 75-85, which restricts access to the cavity, follwed by stabilization of loop [192][193][194][195][196][197][198] and the N-terminal region (residues [5][6][7][8][9][10][11][12][13][14]. These results demonstrated that replacement of a glycine residue by a slightly larger alanine residue did not alter the protein structure significantly, especially as this glycine residue is not involved in substrate binding or catalysis.…”

Protocatechuate 3,4-Dioxygenase: A Wide Substrate Specificity Enzyme Isolated from <b><i>Stenotrophomonas maltophilia</i></b> KB2 as a Useful Tool in Aromatic Acid Biodegradation

“…Interestingly, we observed the high activity of P34O against 2,4-, 2,5-and 3,5-dihydroxybenzoate, substrates which did not have hydroxyl groups in ortho configuration. Typically, the aromatic ring without ortho diol groups is cleaved by gentisate dioxygenase [Chen et al, 2008]. However, the activity of this enzyme was excluded in strain KB2 as it was not able to grow on gentisic acid as a substrate [unpubl.…”