Degradation of benzoate via benzoyl-coenzyme A and gentisate by Bacillus stearothermophilus PK1, and purification of gentisate 1,2-dioxygenase

The thermophilic Bacillus stearothermophilus PK1 utilized benzoate, 3-hydroxybenzoate, and gentisate as sole source of carbon and energy. 2- and 4-Hydroxybenzoate, 2,3- and 3,4-dihydroxybenzoate, and catechol did not support growth. Degradation of benzoate proceeded via benzoyl-coenzyme A (benzoyl-CoA) and gentisate. The inducible benzoyl-CoA ligase converted benzoate but not 3-hydroxybenzoate to its coenzyme A thioester. Gentisate 1,2-dioxygenase from B. stearothermophilus PK1 was purified to homogeneity. The enzyme is presumed to be a homohexamer with a subunit molecular mass of 40 kDa. It showed maximal activity at 65–70°C. After incubation for 80 min at 65°C, 50% of the original activity was lost. Gentisate 1,2-dioxygenase activity from strain PK1 was strictly dependent on exogenously added Fe²⁺, and it was inhibited by metal-chelating agents, indicating an essential role of Fe²⁺ in catalysis.Dedicated to Professor J. C. G. Ottow on the occasion of his 60th birthday