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

Gentisate 1,2-dioxygenase (E.C.1.14.13) was purified to homogeneity from Klebsiella pneumoniae M5aI, 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 mu M for gentisate, Its amino acid content was determined to be very similar to that of the enzyme from Pseudomonas acidovorans.