Anaerobic metabolism of 3-hydroxybenzoate by the denitrifying bacterium Thauera aromatica.
The anaerobic metabolism of 3-hydroxybenzoate was studied in the denitrifying bacterium Thauera aromatica. Cells grown with this substrate were adapted to grow with benzoate but not with 4-hydroxybenzoate. Vice versa, 4-hydroxybenzoate-grown cells did not utilize 3-hydroxybenzoate. The first step in 3-hydroxybenzoate metabolism is a coenzyme A (CoA) thioester formation, which is catalyzed by an inducible 3-hydroxybenzoate-CoA ligase. The enzyme was purified and characterized. Further metabolism of 3-hydroxybenzoyl-CoA by cell extract required MgATP and was coupled to the oxidation of 2 mol of reduced viologen dyes per mol of substrate added. Purification of the 3-hydroxybenzoyl-CoA reducing enzyme revealed that this activity was due to benzoyl-CoA reductase, which reduced the 3-hydroxy analogue almost as efficiently as benzoyl-CoA. The further metabolism of the alicyclic dienoyl-CoA product containing the hydroxyl substitution obviously required additional specific enzymes. Comparison of the protein pattern of 3-hydroxybenzoate-grown cells with benzoate-grown cells revealed several 3-hydroxybenzoate-induced proteins; the N-terminal amino acid sequences of four induced proteins were determined and the corresponding genes were identified and sequenced. A cluster of six adjacent genes contained the genes for substrate-induced proteins 1 to 3; this cluster may not yet be complete. Protein 1 is a short-chain alcohol dehydrogenase. Protein 2 is a member of enoyl-CoA hydratase enzymes. Protein 3 was identified as 3-hydroxybenzoate-CoA ligase. Protein 4 is another member of the enoyl-CoA hydratases. In addition, three genes coding for enzymes of beta-oxidation were present. The anaerobic 3-hydroxybenzoate metabolism here obviously combines an enzyme (benzoyl-CoA reductase) and electron carrier (ferredoxin) of the general benzoyl-CoA pathway with enzymes specific for the 3-hydroxybenzoate pathway. This raises some questions concerning the regulation of both pathways.
Laempe
D
D
Mikrobiologie, Institut für Biologie II, Albert-Ludwigs-Universität, Freiburg, Germany.
Jahn
M
M
Breese
K
K
Schägger
H
H
Fuchs
G
G
D000214:Acyl Coenzyme A / Q000378:metabolism; D000693:Anaerobiosis; D003001:Cloning, Molecular; D003066:Coenzyme A Ligases / Q000302:isolation & purification*; D004790:Enzyme Induction; D005798:Genes, Bacterial; D062385:Hydroxybenzoates / Q000378:metabolism*; D008957:Models, Genetic; D008969:Molecular Sequence Data; D009566:Nitrates / Q000378:metabolism; D009573:Nitrites; D010084:Oxidation-Reduction; D010088:Oxidoreductases / Q000302:isolation & purification / Q000378:metabolism; D044925:Oxidoreductases Acting on CH-CH Group Donors; D017422:Sequence Analysis, DNA; D020539:Sequence Analysis, Protein; D013379:Substrate Specificity; D020628:Thauera / Q000378:metabolism*
C082323:3-hydroxybenzoyl-coenzyme A; D000214:Acyl Coenzyme A; D062385:Hydroxybenzoates; D009566:Nitrates; D009573:Nitrites; C032948:3-hydroxybenzoic acid; D010088:Oxidoreductases; D044925:Oxidoreductases Acting on CH-CH Group Donors; C109030:benzoyl-coenzyme A reductase (dearomatizing); C411162:3-hydroxybenzoate coenzyme A ligase; D003066:Coenzyme A Ligases