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.

Keywords: 2,4′-dihydroxyacetophenone dioxygenase; Alcaligenes sp. 4HAP; limited proteolysis.

Figure 1

The reaction catalysed by 2,4′-dihydroxyacetophenone dioxygenase (DAD). The enzyme has a high affinity for oxygen, which is used for C—C bond cleavage of the substrate (2,4′-dihydroxyacetophenone) yielding 4-hydroxybenzoic acid and formate.

Figure 2

Crystals of DAD that were obtained following limited chymotrypsin digestion. These needle-like crystals are up to 1 mm in length but are around 10 µm in thickness.

Figure 3

The DAD crystal which yielded synchrotron X-ray data to 2.2 Å resolution at the ESRF, Grenoble. The crystal was cryoprotected in 30% glycerol and maintained at −173°C for data collection.

Figure 4

A diffraction image of DAD which was obtained on BM14U at the ESRF. The grey circle indicates a resolution of 2.3 Å and data-processing statistics are shown in Table 1 ▶.