title: Extension of resolution and oligomerization-state studies of 2,4'-dihydroxyacetophenone dioxygenase from Alcaligenes sp. 4HAP.
abstract: The enzyme 2,4'-dihydroxyacetophenone dioxygenase (DAD) catalyses the conversion of 2,4'-dihydroxyacetophenone to 4-hydroxybenzoic acid and formic acid. This enzyme is a very unusual dioxygenase in that it cleaves a C-C bond in a substituent of the aromatic ring rather than within the ring itself. Whilst it has been shown that DAD is a tetramer in solution, the recently solved crystal structure of the Alcaligenes sp. 4HAP enzyme was in fact dimeric rather than tetrameric. Since the use of limited chymotrypsinolysis, which apparently results in removal of the first 20 or so N-terminal residues of DAD, was necessary for crystallization of the protein, it was investigated whether this was responsible for the change in its oligomerization state. Gel-filtration and analytical ultracentrifugation studies were conducted, which confirmed that chymotrypsinolysed DAD has an apparent molecular weight of around 40 kDa, corresponding to a dimer. In contrast, the native enzyme has a molecular weight in the 70-80 kDa region, as expected for the tetramer. The structural basis for tetramerization has been investigated by the use of several docking servers, and the results are remarkably consistent with the tetrameric structure of a homologous cupin protein from Ralstonia eutropha (PDB entry 3ebr).
authors.affiliation: Laboratory of Protein Crystallography, Centre for Amyloidosis and Acute Phase Proteins, UCL Division of Medicine (Royal Free Campus), Rowland Hill Street, London NW2 3PF, England.
authors.forename: J
authors.initials: J
authors.lastname: Guo
authors.affiliation: Laboratory of Protein Crystallography, Centre for Amyloidosis and Acute Phase Proteins, UCL Division of Medicine (Royal Free Campus), Rowland Hill Street, London NW2 3PF, England.
authors.forename: P
authors.initials: P
authors.lastname: Erskine
authors.affiliation: Laboratory of Protein Crystallography, Centre for Amyloidosis and Acute Phase Proteins, UCL Division of Medicine (Royal Free Campus), Rowland Hill Street, London NW2 3PF, England.
authors.forename: A R
authors.initials: AR
authors.lastname: Coker
authors.affiliation: Department of Structural and Molecular Biology, Division of Biosciences, University College London, Darwin Building, Gower Street, London WC1E 6BT, England.
authors.forename: J
authors.initials: J
authors.lastname: Gor
authors.affiliation: Department of Structural and Molecular Biology, Division of Biosciences, University College London, Darwin Building, Gower Street, London WC1E 6BT, England.
authors.forename: S J
authors.initials: SJ
authors.lastname: Perkins
authors.affiliation: Laboratory of Protein Crystallography, Centre for Amyloidosis and Acute Phase Proteins, UCL Division of Medicine (Royal Free Campus), Rowland Hill Street, London NW2 3PF, England.
authors.forename: S P
authors.initials: SP
authors.lastname: Wood
authors.affiliation: Laboratory of Protein Crystallography, Centre for Amyloidosis and Acute Phase Proteins, UCL Division of Medicine (Royal Free Campus), Rowland Hill Street, London NW2 3PF, England.
authors.forename: J B
authors.initials: JB
authors.lastname: Cooper
mesh_terms: D000421:Alcaligenes / Q000201:enzymology*; D055162:Biocatalysis; D002850:Chromatography, Gel; D003460:Crystallization; D018360:Crystallography, X-Ray; D049308:Dioxygenases / Q000737:chemistry*; D008958:Models, Molecular; D055503:Protein Multimerization; D020836:Protein Structure, Quaternary; D055672:Static Electricity; D014461:Ultracentrifugation
chemical_list: D049308:Dioxygenases; C403717:2,4'-dihydroxyacetophenone dioxygenase
keywords: 2,4′-dihydroxyacetophenone dioxygenase; analytical ultracentrifugation; dioxygenase; docking; gel filtration; oligomerization; protein X-ray structure