Molecular and Catalytic Properties of 2,4′-Dihydroxyacetophenone Dioxygenase from Burkholderia sp. AZ11

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 (M _r=90 kDa) was a homotetramer with a subunit M _r 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 K _m for DHAP and the apparent V _max 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.

• Burkholderia
• dihydroxyacetophenone
• dioxygenase
• metalloenzyme