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Crystallization and preliminary X-ray characterization of the 2,4'-dihydroxyacetophenone dioxygenase from Alcaligenes sp. 4HAP.

ABSTRACT: The enzyme 2,4'-dihydroxyacetophenone dioxygenase (or DAD) catalyses the conversion of 2,4'-dihydroxyacetophenone to 4-hydroxybenzoic acid and formic acid with the incorporation of molecular oxygen. Whilst the vast majority of dioxygenases cleave within the aromatic ring of the substrate, DAD is very unusual in that it is involved in C-C bond cleavage in a substituent of the aromatic ring. There is evidence that the enzyme is a homotetramer of 20.3?kDa subunits each containing nonhaem iron and its sequence suggests that it belongs to the cupin family of dioxygenases. By the use of limited chymotrypsinolysis, the DAD enzyme from Alcaligenes sp. 4HAP has been crystallized in a form that diffracts synchrotron radiation to a resolution of 2.2?Å.

SUBMITTER: Beaven G

PROVIDER: S-EPMC4051546 | biostudies-literature | 2014 Jun

REPOSITORIES: biostudies-literature

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Crystallization and preliminary X-ray characterization of the 2,4'-dihydroxyacetophenone dioxygenase from Alcaligenes sp. 4HAP.

Beaven G G Bowyer A A Erskine P P Wood S P SP McCoy A A Coates L L Keegan R R Lebedev A A Hopper D J DJ Kaderbhai M A MA Cooper J B JB

Acta crystallographica. Section F, Structural biology communications 20140525 Pt 6

The enzyme 2,4'-dihydroxyacetophenone dioxygenase (or DAD) catalyses the conversion of 2,4'-dihydroxyacetophenone to 4-hydroxybenzoic acid and formic acid with the incorporation of molecular oxygen. Whilst the vast majority of dioxygenases cleave within the aromatic ring of the substrate, DAD is very unusual in that it is involved in C-C bond cleavage in a substituent of the aromatic ring. There is evidence that the enzyme is a homotetramer of 20.3 kDa subunits each containing nonhaem iron and i ...[more]

PMID: 24915102

Similar Datasets

Structure of the 2,4'-dihydroxyacetophenone dioxygenase from Alcaligenes sp. 4HAP.

Project description:The enzyme 2,4'-dihydroxyacetophenone dioxygenase (DAD) catalyses the conversion of 2,4'-dihydroxyacetophenone to 4-hydroxybenzoic acid and formic acid with the incorporation of molecular oxygen. Whilst the vast majority of dioxygenases cleave within the aromatic ring of the substrate, DAD is very unusual in that it is involved in C-C bond cleavage in a substituent of the aromatic ring. There is evidence that the enzyme is a homotetramer of 20.3 kDa subunits, each containing nonhaem iron, and its sequence suggests that it belongs to the cupin family of dioxygenases. In this paper, the first X-ray structure of a DAD enzyme from the Gram-negative bacterium Alcaligenes sp. 4HAP is reported, at a resolution of 2.2 Å. The structure establishes that the enzyme adopts a cupin fold, forming dimers with a pronounced hydrophobic interface between the monomers. The catalytic iron is coordinated by three histidine residues (76, 78 and 114) within a buried active-site cavity. The iron also appears to be tightly coordinated by an additional ligand which was putatively assigned as a carbonate dianion since this fits the electron density optimally, although it might also be the product formate. The modelled carbonate is located in a position which is highly likely to be occupied by the α-hydroxyketone group of the bound substrate during catalysis. Modelling of a substrate molecule in this position indicates that it will interact with many conserved amino acids in the predominantly hydrophobic active-site pocket where it undergoes peroxide radical-mediated heterolysis.

| S-EPMC4219425 | biostudies-literature

Extension of resolution and oligomerization-state studies of 2,4'-dihydroxyacetophenone dioxygenase from Alcaligenes sp. 4HAP.

Project description: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).

| S-EPMC4601589 | biostudies-literature

2,4'-Dihydroxyacetophenone dioxygenase (EC 1.13.11.41) from Alcaligenes sp. 4HAP: a novel enzyme with an atypical dioxygenase sequence.

Project description:2,4'-Dihydroxyacetophenone dioxygenase (EC 1.13.11.41) was purified to homogeneity from Alcaligenes sp. 4HAP grown on 4-hydroxyacetophenone. Measurements of the M(r) of the native enzyme ranged from 81600 to 87000, whereas values of 21000 and 20379 were given by SDS/PAGE and electrospray MS respectively. The enzyme is a homotetramer and contains one atom of iron per molecule of enzyme. From C- and N-terminal analyses, primers for PCR were designed and the dad gene cloned and sequenced. The predicted amino acid sequence of dad, deduced from the nucleotide sequence, confirms the N-terminal amino acid sequencing data and contains the sequence of an internal tryptic peptide. It gave a calculated M(r) of 20364. The gene was expressed in Escherichia coli and yielded active enzyme. The derived amino acid sequence does not show significant similarity to other dioxygenases or any strong similarity to protein sequences presently available in the databases.

| S-EPMC1220656 | biostudies-other

The metal- and substrate-dependences of 2,4'-dihydroxyacetophenone dioxygenase.

Project description:While the enzyme, 2,4'-dihydroxyacetophenone dioxygenase (DAD), has been known for decades, very little has been characterized of the mechanism of the DAD-catalyzed oxidative cleavage of its reported substrate, 2,4'-dihydroxyacetophenone (DHA). The purpose of this study was to identify the active metal center and to characterize the substrate-dependence of the kinetics of the reaction to lay the foundation for deeper mechanistic investigation. To this, the DAD V1M mutant (bDAD) was overexpressed, purified, and reconstituted with various metal ions. Kinetic assays evaluating the activity of the reconstituted enzyme as well as the substrate- and product-dependences of the reaction kinetics were performed. The results from reconstitution of the apoprotein with a variety of metal ions support the requirement for an Fe3+ center for enzyme activity. Reaction rates showed simple saturation kinetics for DHA with values for kcat and KDHA of 2.4 s-1 and 0.7 μM, respectively, but no significant dependence on the concentration of O2. A low-level inhibition (KI = 1100 μM) by the 4HB product was observed. The results support a minimal kinetic model wherein DHA binds to resting ferric enzyme followed by rapid addition of O2 to yield an intermediate complex that irreversibly collapses to products.

| S-EPMC7484352 | biostudies-literature

Crystallization and preliminary X-ray crystallographic analysis of hydroquinone dioxygenase from Sphingomonas sp. TTNP3.

Project description:Hydroquinone dioxygenase (HQDO), a novel Fe(II) ring-fission dioxygenase from Sphingomonas sp. strain TTNP3 which oxidizes a wide range of hydroquinones to the corresponding 4-hydroxymuconic semialdehydes, has been crystallized. The enzyme is an ?(2)?(2) heterotetramer constituted of two subunits of 19 and 38?kDa. Diffraction-quality crystals of HQDO were obtained using the sitting-drop vapour-diffusion method at 277?K from a solution consisting of 16% PEG 4000, 0.3?M MgCl(2), 0.1?M Tris pH 8.5. The crystals belonged to the monoclinic space group P2(1), with unit-cell parameters a = 88.4, b = 125.4, c = 90.8?Å, ? = 105.3°. The asymmetric unit contained two heterotetramers, i.e. four copies of each of the two different subunits related by noncrystallographic 222 symmetry. A complete data set extending to a maximum resolution of 2.5?Å was collected at 100?K using a wavelength of 0.980?Å.

| S-EPMC3374519 | biostudies-literature

Crystallization and preliminary X-ray diffraction experiments of arylmalonate decarboxylase from Alcaligenes bronchisepticus.

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| S-EPMC2443963 | biostudies-literature

Crystallization and preliminary X-ray diffraction studies of a ferredoxin reductase component of carbazole 1,9a-dioxygenase from Novosphingobium sp. KA1.

Project description:Carbazole 1,9a-dioxygenase (CARDO) is the initial enzyme of the carbazole-degradation pathway. The CARDO of Novosphingobium sp. KA1 consists of a terminal oxygenase, a putidaredoxin-type ferredoxin and a ferredoxin-NADH oxidoreductase (Red) and is classified as a class IIA Rieske oxygenase. Red from KA1 was crystallized at 278 K by the hanging-drop vapour-diffusion method using PEG 4000. The crystal diffracted to 1.58 A resolution and belonged to space group P3(2), with unit-cell parameters a = b = 92.2, c = 78.6 A, alpha = gamma = 90, beta = 120 degrees . Preliminary analysis of the X-ray diffraction data revealed that the asymmetric unit contained two Red monomers. The crystal appeared to be a merohedral twin, with a twin fraction of 0.32 and twin law (-h, -k, l).

| S-EPMC2882777 | biostudies-literature

Isolation and preliminary characterization of a o-nitrobenzaldehyde-degrading Alcaligenes sp. ND1.

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| S-EPMC3768461 | biostudies-literature

Crystallization and preliminary crystallographic analysis of gallate dioxygenase DesB from Sphingobium sp. SYK-6.

Project description:Gallate dioxygenase (DesB) from Sphingobium sp. SYK-6, which belongs to the type II extradiol dioxygenase family, was purified and crystallized using the hanging-drop vapour-diffusion method. Two crystal forms were obtained. The form I crystal belonged to space group C2, with unit-cell parameters a = 136.2, b = 53.6, c = 55.1 angstrom, beta = 112.8 degrees, and diffracted to 1.6 angstrom resolution. The form II crystal belonged to space group P2(1), with unit-cell parameters a = 56.2, b = 64.7, c = 116.1 angstrom, beta = 95.1 degrees, and diffracted to 1.9 angstrom resolution. A molecular-replacement calculation using LigAB as a search model yielded a satisfactory solution for both crystal forms.

| S-EPMC2777051 | biostudies-literature

Crystallization and preliminary X-ray diffraction analysis of recombinant chlorocatechol 1,2-dioxygenase from Pseudomonas putida.

Project description:Chlorocatechol 1,2-dioxygenase from the Gram-negative bacterium Pseudomonas putida (Pp 1,2-CCD) is considered to be an important biotechnological tool owing to its ability to process a broad spectrum of organic pollutants. In the current work, the crystallization, crystallographic characterization and phasing of the recombinant Pp 1,2-CCD enzyme are described. Reddish-brown crystals were obtained in the presence of polyethylene glycol and magnesium acetate by utilizing the vapour-diffusion technique in sitting drops. Crystal dehydration was the key step in obtaining data sets, which were collected on the D03B-MX2 beamline at the CNPEM/MCT - LNLS using a MAR CCD detector. Pp 1,2-CCD crystals belonged to space group P6(1)22 and the crystallographic structure of Pp 1,2-CCD has been solved by the MR-SAD technique using Fe atoms as scattering centres and the coordinates of 3-chlorocatechol 1,2-dioxygenase from Rhodococcus opacus (PDB entry 2boy) as the search model. The initial model, which contains three molecules in the asymmetric unit, has been refined to 3.4 Å resolution.

| S-EPMC3080162 | biostudies-literature

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