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The structure of catechol 1,2 dioxygenase from the Acinetobacter sp. ADP1 (Ac 1,2-CTD) has been solved to 2.0 Å. The structure of Ac 1,2-CTD was also solved in complex with two substrates, catechol and 4-methylcatechol, at 1.9 Å and 1.8 Å resolution respectively. Ac 1,2-CTD is an archetypical intradiol dioxygenase and catalyzes the cleavage of catechol concomitant with the incorporation of molecular oxygen into the product cis,cis-muconic acid. The enzyme was overexpressed and purified from an Escherichia coli clone to a specific activity of 16u/mg. Ac 1,2-CTD crystallized at a protein concentration of 60mg/ml in 15%Peg5000, .2M MgAcetate, and 100mM Tris-HCl pH 7.5 using hanging drop vapor diffusion. The crystals belong to the monoclinic space group P2(1) with cell dimensions a=52.6, b=87.5, c=84.2 and ß=96.25. Residues 110-320 constitute a catalytic domain which has significant sequence homology and high structural similarity to another intradiol dioxygenase - protocatechuate 3,4-dioxygenase (3,4-PCD). This domain in Ac 1,2-CTD and PCD is a ß-sandwich composed of two mixed stranded ß-sheets surrounded by several large loops and small helices. All of the ligands to the catalytic non-haem ferric iron originate from this domain. As in 3,4-PCD the active site is coordinated by two axial ligands Y200 and H226 - and three equatorial ligands Y164, H224 and a water molecule in a distorted trigonal bipyramidal geometry. As seen in 3,4-PCD substrate binding leads to the dissociation of Y200, as one hydroxyl of substrate binds in an axial position and one binds in an equitoral position. A major difference between 3,4-PCD and Ac 1,2-CTD is that unlike 3,4-PCD all the residues that form the active site originate from one subunit. Despite this dissimilarity several residues which have no corresponding residue in sequence alignments do have structurally related homologs originating from an altogether different part of the sequence. A series of 5 extended helices (residues 1-110) from each subunit intertwine with one another to form a helical zipper linking the two catalytic domains together. Unexpectedly the helices form a 8x35 Å channel which are shown to bind two phospholipid like molecules. The possible function of this linker domain and the bound phospholipids is discussed. |
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