Purification and biochemical characterization of gentisate 1,2-dioxygenase from Klebsiella pneumoniae M5a1

Abstract

Gentisate 1,2-dioxygenase (E.C.1.14.13) was purified to homogeneity from Klebsiella pneumoniae M5a1, a soil bacterium able to degrade a great variety of aromatic compounds. The molecular mass of the purified holoenzyme was 159 kDa and its structure was deduced to be a tetramer with 38 kDa per subunit. Gentisate 1,2-dioxygenase appears to contain Fe2+ in its active site. The optimum temperature for enzyme activity was estimated to be 30 degrees C, the optimum pH values varied between 8 and 9 and the isoelectric point was 4.7. Gentisate dioxygenase exhibited typical saturation kinetics and had an apparent K(m) of 52 microM for gentisate. Its amino acid content was determined to be very similar to that of the enzyme from Pseudomonas acidovorans.