Crystallization and preliminary X-ray characterization of the 2,4′-dihydroxyacetophenone dioxygenase from<i>Alcaligenes</i>sp. 4HAP

Beaven, G. D. E.; Bowyer, A.; Erskine, P.T.; Wood, Steve; McCoy, Airlie J.; Coates, Leighton; Keegan, Ronan; Лебедев, А. А.; Hopper, David J.; Kaderbhai, Mustak A.; Cooper, J.B.

doi:10.1107/s2053230x14009649

Cited by 4 publications

(8 citation statements)

References 14 publications

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“…Data-processing statistics are shown in Table 1, where it can be seen that the traditional R merge is slightly high at 13.6% and the outer shell completeness is slightly low, but that the data are of excellent consistency to 2.2 Å resolution according to the half-set correlation criterion of Karplus & Diederichs (2012). A more comprehensive account of the expression, purification, crystallization and data collection is given in Beaven et al (2014).…”

Section: Methodsmentioning

confidence: 99%

“…Crystals of Alcaligenes sp. 4HAP DAD which diffracted synchrotron radiation to 2.2 Å resolution were obtained by in situ chymotrypsinolysis (Beaven et al, 2014). They belonged to space group P6 5 22 with one molecule per asymmetric unit and allowed the structure to be solved and refined to an R factor of 17.3% and an R free of 21.8%.…”

Section: Crystallographic Datamentioning

confidence: 99%

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Structure of the 2,4′-dihydroxyacetophenone dioxygenase fromAlcaligenessp. 4HAP

Keegan

Lebedev

Erskine

et al. 2014

Acta Cryst D Biol Crystallogr

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The enzyme 2,4 0 -dihydroxyacetophenone dioxygenase (DAD) catalyses the conversion of 2,4 0 -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 Gramnegative 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 -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.

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Section: Methodsmentioning

confidence: 99%

Section: Crystallographic Datamentioning

confidence: 99%

Structure of the 2,4′-dihydroxyacetophenone dioxygenase fromAlcaligenessp. 4HAP

Keegan

Lebedev

Erskine

et al. 2014

Acta Cryst D Biol Crystallogr

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“…Use of the MATT-PROB server (Kantardjieff & Rupp, 2003) indicated that there were likely to be two molecules of DAD (which are approximately 20 kDa each) in the asymmetric unit. However, attempts at molecular replacement with various search models which had sequence homology to DAD failed to yield a solution, and when the structure of DAD was solved in a different crystal form (Beaven et al, 2014;Keegan et al, 2014) this also did not provide a convincing solution with the 1.1 Å resolution data set.…”

Section: Data Processingmentioning

confidence: 99%

The 1.1 Å resolution structure of a periplasmic phosphate-binding protein fromStenotrophomonas maltophilia: a crystallization contaminant identified by molecular replacement using the entire Protein Data Bank

Keegan

Hopper

Coates

et al. 2016

Acta Crystallogr D Struct Biol

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During efforts to crystallize the enzyme 2,4-dihydroxyacetophenone dioxygenase (DAD) from Alcaligenes sp. 4HAP, a small number of strongly diffracting protein crystals were obtained after two years of crystal growth in one condition. The crystals diffracted synchrotron radiation to almost 1.0 Å resolution and were, until recently, assumed to be formed by the DAD protein. However, when another crystal form of this enzyme was eventually solved at lower resolution, molecular replacement using this new structure as the search model did not give a convincing solution with the original atomic resolution data set. Hence, it was considered that these crystals might have arisen from a protein impurity, although molecular replacement using the structures of common crystallization contaminants as search models again failed. A script to perform molecular replacement using MOLREP in which the first chain of every structure in the PDB was used as a search model was run on a multi-core cluster. This identified a number of prokaryotic phosphate-binding proteins as scoring highly in the MOLREP peak lists. Calculation of an electron-density map at 1.1 Å resolution based on the solution obtained with PDB entry 2q9t allowed most of the amino acids to be identified visually and built into the model. A BLAST search then indicated that the molecule was most probably a phosphate-binding protein from Stenotrophomonas maltophilia (UniProt ID B4SL31; gene ID Smal_2208), and fitting of the corresponding sequence to the atomic resolution map fully corroborated this. Proteins in this family have been linked to the virulence of antibiotic-resistant strains of pathogenic bacteria and with biofilm formation. The structure of the S. maltophilia protein has been refined to an R factor of 10.15% and an Rfree of 12.46% at 1.1 Å resolution. The molecule adopts the type II periplasmic binding protein (PBP) fold with a number of extensively elaborated loop regions. A fully dehydrated phosphate anion is bound tightly between the two domains of the protein and interacts with conserved residues and a number of helix dipoles.

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“…4HAP has been shown to be a homotetramer in which each subunit has a molecular mass of 20.3 kDa and consists of 177 amino acids (Hopper & Kaderbhai, 1999). Recently, structural studies have shown that it is a member of the cupin superfamily, which is characterized by a conserved -barrel fold (Beaven et al, 2014;Keegan et al, 2014). The core cupin fold of DAD is formed by residues 60-132, which assemble into two four-stranded antiparallel -sheets, forming a double-stranded -helix that is extended at one end by an additional -hairpin in each sheet.…”

Section: Introductionmentioning

confidence: 99%

“…The putative binding site for the aromatic substrate is buried and is mainly composed of conserved hydrophobic residues. The two -hairpins forming one end of the barrel are somewhat splayed apart, exposing an unusual hydrophobic patch which might be the entry point for substrate (Beaven et al, 2014;Keegan et al, 2014).…”

Section: Introductionmentioning

confidence: 99%

Extension of resolution and oligomerization-state studies of 2,4′-dihydroxyacetophenone dioxygenase fromAlcaligenessp. 4HAP

et al. 2015

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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).

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Crystallization and preliminary X-ray characterization of the 2,4′-dihydroxyacetophenone dioxygenase fromAlcaligenessp. 4HAP

Cited by 4 publications

References 14 publications

Structure of the 2,4′-dihydroxyacetophenone dioxygenase fromAlcaligenessp. 4HAP

Structure of the 2,4′-dihydroxyacetophenone dioxygenase fromAlcaligenessp. 4HAP

The 1.1 Å resolution structure of a periplasmic phosphate-binding protein fromStenotrophomonas maltophilia: a crystallization contaminant identified by molecular replacement using the entire Protein Data Bank

Extension of resolution and oligomerization-state studies of 2,4′-dihydroxyacetophenone dioxygenase fromAlcaligenessp. 4HAP

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