Purification and properties of homoprotocatechuate 2,3-dioxygenase from Bacillus stearothermophilus.

The enzyme 3,4-dihydroxyphenylacetate:oxygen 2,3-oxidoreductase (decyclizing) (homoprotocatechuate 2,3-dioxygenase) was purified from the thermophilic organism Bacillus stearothermophilus, grown with j-hydroxyphenylacetic acid as a source of carbon. The enzyme appeared to be homogeneous as judged by disc-gel electrophoresis and sedimentation equilibrium measurements. The average molecular weight determined by three independent procedures was 106,000; the protein was globular and was dissociated in sodium dodecyl sulfate to give a species of molecular weight 33,000 to 35,000. The enzyme was fairly stable on heating and showed maximal activity at about 57 degrees C. An Arrhenius plot of Km for homoprotocatechuate was concave upward, with a break at 32 degrees C; an increase in delta H above this temperature was compensated by lower values of --delta S. Several properties of this enzyme are contrasted with those reported for homoprotocatechuate 2,3-dioxygenase purified by other workers from Pseudomonas ovalis.