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The structure of homoprotocatechuate 2,3-dioxygenase from Brevibacterium fuscum (Bf 2,3-HPCD) has been determined to 1.6 Å resolution. Bf 2,3-HPCD is a member of a family of enzymes which catalyze the extradiol cleavage of catecholate substrates into the corresponding muconic semialdehydes. Mechanistically the reaction is proposed to occur through the activation of dioxygen by ferrous iron, leading to nucleophilic attack by the emerging superoxide on the catecholic ring. Bf 2,3-HPCD was overexpressed and purified from an Escherichia coli clone to a specific activity of 15U/mg. The enzyme was crystallized in three space groups P321, C2 and I4(1). A limited proteolysis with trypsin (~41 residues) was required to obtain the C2 and I4(1) crystal form. This removed a small fragment which appeared to have high mobility and was interfering with crystallization. Bf 2,3-HPCD and HPCD from Arthrobacter globiformus (Ag 2,3-HPCD) are the only extradiol dioxygenases with this high mobility C-TERM fragment. Using the I4(1) crystal form a combination of single isomorphous replacement, solvent flattening, and four fold non-crystallographic averaging was used to obtain phases adequate for modeling the data. A high resolution data set was collected using synchrotron radiation at Argonne National Laboratories to extend the resolution to 1.6 Å. Bf 2,3-HPCD is a homotetramer of identical subunits. Each subunit is composed of two domains ( N-terminal, C-terminal). Each of these domains is made of two structurally homologous ßaßßß motifs which combine to form a ß-barrel. The C-terminal domain ß-barrel supplies all the ligands to the ferrous iron and makes up the residues of the active site. Despite having less than 25% sequence homology, Bf 2,3-HPCD has the same fold and active site ligands seen in the recently determined structures of 2,3-dihydroxybiphenyl 1,2-dioxygenase, and catechol 2,3-dioxygenase. The active site iron is coordinated by two axial ligands H214, and a water molecule - and four equatorial ligands H155, E267 and three water molecules in an octahedral geometry. Several residues around the active site are structurally conserved (H200, H248, Y257) and are most likely important for the mechanism, while others are involved in the unique substrate specificity of Bf 2,3-HPCD (R243, R292, R293, W192, W304). Molecular replacement was used to solve the P321 crystal form of Bf 2,3-HPCD. The protein in this crystal form was not treated with trypsin so it was thought the C-TERM mobile fragment may be visible. Indeed electron density was apparent for the C-TERM fragment and was fitted with the remaining 40+ residues.. What was originally thought to be a functionless mobile fragment has turned out to be an interesting caviate in the binding of substrate; and possibly the stabilization of the active site metal in this subfamily of extradiol dioxygenases. |
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