Barbara Tshisuaka

Bacterial Degradation of Quinoline and Derivatives—Pathways and Their Biocatalysts

A series of interesting enzymes were discovered during investigations on the degradation of quinoline by microorganisms. These include the molybdenum-containing hydroxylases that catalyze the transformation 1→2 and the unusual 2,4-dioxygenases that catalyze the reaction 3→4. The application of the hydroxylases may even be interesting in industry, because several quinoline derivatives are used as pharmaceuticals or agrochemicals.

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.

Molecular cloning, sequencing, expression, and site-directed mutagenesis of the 1H-3-hydroxy-4-oxoquinaldine 2,4-dioxygenase gene fromArthrobacter spec. R?61a

The ring cleaving enzyme 1H‐3‐hydroxy‐4‐oxoquinaldine 2,4‐dioxygenase (HOD) of Arthrobacter spec. R?61a is part of the quinaldine degradation pathway. Carbon monoxide and N‐acetyl‐anthranilate are the products formed by dioxygenolytic cleavage of two C—C bonds in the substrates pyridine ring. The gene coding for HOD was cloned and sequenced. An isoelectric point of pH 5.40 and a molecular mass of 31,838 Da was deduced from the sequence. HOD is shown to be remarkably similar to 1H‐3‐hydroxy‐4‐oxoquinoline 2,4‐dioxygenase (QDO) of Pseudomonas putida 33/1, but not to other dioxygenases described so far. Consensus regions indicative for any chromophoric cofactor or any catalytically relevant metal were not detected. Sequence comparisons and secondary structure predictions revealed HOD as a new member of the α/β hydrolase fold family. Expression in E. coli yielded recombinant catalytically active His‐tagged HOD. S101A and D233A, two mutants of HOD, were obtained by site‐directed mutagenesis. Since their residual activity is 43.1% and 62.6%, respectively, they probably are of no catalytic relevance although they might play a role in the interaction between enzyme and substrate.