The toxicity of acetylacetone has been demonstrated in various studies. Little is known, however, about metabolic pathways for its detoxification or mineralization. Data presented here describe for the first time the microbial degradation of acetylacetone and the characterization of a novel enzyme that initiates the metabolic pathway. From an Acinetobacter johnsonii strain that grew with acetylacetone as the sole carbon source, an inducible acetylacetone-cleaving enzyme was purified to homogeneity. The corresponding gene, coding for a 153 amino acid sequence that does not show any significant relationship to other known protein sequences, was cloned and overexpressed in Escherichia coli and gave high yields of active enzyme. The enzyme cleaves acetylacetone to equimolar amounts of methylglyoxal and acetate, consuming one equivalent of molecular oxygen. No exogenous cofactor is required, but Fe2+ is bound to the active protein and essential for its catalytic activity. The enzyme has a high affinity for acetylacetone with a Km of 9.1μM and a kcat of 8.5s-1. A metabolic pathway for acetylacetone degradation and the putative relationship of this novel enzyme to previously described dioxygenases are discussed.
- acetylacetone degradation pathway
- dicarbonyl cleavage
- diketone cleavage
- iron cofactor
↵2 Present address: Research Center Borstel, D-23845 Borstel, Germany.
The nucleotide sequence data for open reading frame ORF2 are available at the GenBank™ Nucleotide Sequence Database under accession number AF489107.
Abbreviations used: MALDI-TOF MS, matrix-assisted laser-desorption ionization—time-of-flight MS; nDke1, native diketone-cleaving enzyme; rDke1, recombinant diketone-cleaving enzyme.
- The Biochemical Society, London ©2003