Abstract
The gene dad encoding 2,4′-dihydroxyacetophenone (DHAP) dioxygenase was cloned from Burkholderia sp. AZ11. The initiation codon GTG was converted to ATG for high-level expression of the enzyme in Escherichia coli. The enzyme was moderately thermostable, and the recombinant enzyme was briefly purified. The enzyme (Mr=90 kDa) was a homotetramer with a subunit Mr of 23 kDa. It contained 1.69 mol of non-heme iron, and had a dark gray color. On anaerobic incubation of it with DHAP, the absorption at around 400 nm increased due to the formation of an enzyme-DHAP complex. Multiple sequence alignment suggested that His77, His79, His115, and Glu96 in the cupin fold were possible metal ligands. The apparent Km for DHAP and the apparent Vmax were estimated to be 1.60 μM and 6.28 μmol/min/mg respectively. 2-Hydroxyacetophenone was a poor substrate. CuCl2 and HgCl2 strongly inhibited the enzyme, while FeSO4 weakly activated it.
