Anaerobic metabolism of l -phenylalanine via benzoyl-CoA in the denitrifying bacterium Thauera aromatica

1997 ◽  
Vol 168 (4) ◽  
pp. 310-320 ◽  
Author(s):  
Sabine Schneider ◽  
M. E.-S. Mohamed ◽  
G. Fuchs
Keyword(s):  
Anaerobic Metabolism ◽  
Denitrifying Bacterium ◽  
Thauera Aromatica

scholarly journals Anaerobic Metabolism of 3-Hydroxybenzoate by the Denitrifying Bacterium Thauera aromatica

2001 ◽  
Vol 183 (3) ◽  
pp. 968-979 ◽  
Author(s):  
Diana Laempe ◽  
Martina Jahn ◽  
Klaus Breese ◽  
Hermann Schägger ◽  
Georg Fuchs
Keyword(s):  
Anaerobic Metabolism ◽  
Protein Pattern ◽  
Cell Extract ◽  
Amino Acid Sequences ◽  
Denitrifying Bacterium ◽  
Thauera Aromatica ◽  
Coa Ligase ◽  
Complete Protein ◽  
Coa Reductase ◽  
Induced Proteins

ABSTRACT 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 β-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.

Differential induction of enzymes involved in anaerobic metabolism of aromatic compounds in the denitrifying bacterium Thauera aromatica

1998 ◽  
Vol 170 (2) ◽  
pp. 120-131 ◽  
Author(s):  
J. Heider ◽  
Matthias Boll ◽  
Klaus Breese ◽  
Sabine Breinig ◽  
Christa Ebenau-Jehle ◽  
...  
Keyword(s):  
Aromatic Compounds ◽  
Anaerobic Metabolism ◽  
Denitrifying Bacterium ◽  
Thauera Aromatica ◽  
Induction Of Enzymes ◽  
Differential Induction

scholarly journals Anaerobic Metabolism of Catechol by the Denitrifying Bacterium Thauera aromatica—a Result of Promiscuous Enzymes and Regulators?

2007 ◽  
Vol 190 (5) ◽  
pp. 1620-1630 ◽  
Author(s):  
Bin Ding ◽  
Sirko Schmeling ◽  
Georg Fuchs
Keyword(s):  
Electron Acceptor ◽  
Anaerobic Metabolism ◽  
Regulatory Proteins ◽  
Hydroxyl Group ◽  
Denitrifying Bacterium ◽  
Aromatic Substrates ◽  
Thauera Aromatica ◽  
Coa Ligase ◽  
Coa Reductase ◽  
Lines Of Evidence

ABSTRACT The anaerobic metabolism of catechol (1,2-dihydroxybenzene) was studied in the betaproteobacterium Thauera aromatica that was grown with CO2 as a cosubstrate and nitrate as an electron acceptor. Based on different lines of evidence and on our knowledge of enzymes and genes involved in the anaerobic metabolism of other aromatic substrates, the following pathway is proposed. Catechol is converted to catechylphosphate by phenylphosphate synthase, which is followed by carboxylation by phenylphosphate carboxylase at the para position to the phosphorylated phenolic hydroxyl group. The product, protocatechuate (3,4-dihydroxybenzoate), is converted to its coenzyme A (CoA) thioester by 3-hydroxybenzoate-CoA ligase. Protocatechuyl-CoA is reductively dehydroxylated to 3-hydroxybenzoyl-CoA, possibly by 4-hydroxybenzoyl-CoA reductase. 3-Hydroxybenzoyl-CoA is further metabolized by reduction of the aromatic ring catalyzed by an ATP-driven benzoyl-CoA reductase. Hence, the promiscuity of several enzymes and regulatory proteins may be sufficient to create the catechol pathway that is made up of elements of phenol, 3-hydroxybenzoate, 4-hydroxybenzoate, and benzoate metabolism.

scholarly journals Phenylphosphate Synthase: a New Phosphotransferase Catalyzing the First Step in Anaerobic Phenol Metabolism in Thauera aromatica

2004 ◽  
Vol 186 (23) ◽  
pp. 8044-8057 ◽  
Author(s):  
Sirko Schmeling ◽  
Ariun Narmandakh ◽  
Oliver Schmitt ◽  
Nasser Gad'on ◽  
Karola Schühle ◽  
...  
Keyword(s):  
Anaerobic Metabolism ◽  
Atp Hydrolysis ◽  
Second Step ◽  
Phosphoryl Group ◽  
The Novel ◽  
Thauera Aromatica ◽  
Phosphoenol Pyruvate ◽  
Pyruvate Synthase ◽  
Phosphoenolpyruvate Synthase ◽  
Phenol Moiety

ABSTRACT The anaerobic metabolism of phenol in the beta-proteobacterium Thauera aromatica proceeds via para-carboxylation of phenol (biological Kolbe-Schmitt carboxylation). In the first step, phenol is converted to phenylphosphate which is then carboxylated to 4-hydroxybenzoate in the second step. Phenylphosphate formation is catalyzed by the novel enzyme phenylphosphate synthase, which was studied. Phenylphosphate synthase consists of three proteins whose genes are located adjacent to each other on the phenol operon and were overproduced in Escherichia coli. The promoter region and operon structure of the phenol gene cluster were investigated. Protein 1 (70 kDa) resembles the central part of classical phosphoenolpyruvate synthase which contains a conserved histidine residue. It catalyzes the exchange of free [14C]phenol and the phenol moiety of phenylphosphate but not the phosphorylation of phenol. Phosphorylation of phenol requires protein 1, MgATP, and another protein, protein 2 (40 kDa), which resembles the N-terminal part of phosphoenol pyruvate synthase. Proteins 1 and 2 catalyze the following reaction: phenol + MgATP + H2O→phenylphosphate + MgAMP + orthophosphate. The phosphoryl group in phenylphosphate is derived from the β-phosphate group of ATP. The free energy of ATP hydrolysis obviously favors the trapping of phenol (Km , 0.04 mM), even at a low ambient substrate concentration. The reaction is stimulated severalfold by another protein, protein 3 (24 kDa), which contains two cystathionine-β-synthase domains of unknown function but does not show significant overall similarity to known proteins. The molecular and catalytic features of phenylphosphate synthase resemble those of phosphoenolpyruvate synthase, albeit with interesting modifications.

scholarly journals Benzoate-Coenzyme A Ligase from Thauera aromatica: an Enzyme Acting in Anaerobic and Aerobic Pathways

2003 ◽  
Vol 185 (16) ◽  
pp. 4920-4929 ◽  
Author(s):  
Karola Schühle ◽  
Johannes Gescher ◽  
Ulrich Feil ◽  
Michael Paul ◽  
Martina Jahn ◽  
...  
Keyword(s):  
Anaerobic Metabolism ◽  
Coenzyme A ◽  
Aromatic Acids ◽  
Aerobic Growth ◽  
Aromatic Substrates ◽  
Thauera Aromatica ◽  
Coa Ligase ◽  
Anaerobic Aromatic Metabolism ◽  
Coenzyme A Ligase ◽  
Specificity Constant

ABSTRACT In the denitrifying member of the β-Proteobacteria Thauera aromatica, the anaerobic metabolism of aromatic acids such as benzoate or 2-aminobenzoate is initiated by the formation of the coenzyme A (CoA) thioester, benzoyl-CoA and 2-aminobenzoyl-CoA, respectively. Both aromatic substrates were transformed to the acyl-CoA intermediate by a single CoA ligase (AMP forming) that preferentially acted on benzoate. This benzoate-CoA ligase was purified and characterized as a 57-kDa monomeric protein. Based on V max/Km , the specificity constant for 2-aminobenzoate was 15 times lower than that for benzoate; this may be the reason for the slower growth on 2-aminobenzoate. The benzoate-CoA ligase gene was cloned and sequenced and was found not to be part of the gene cluster encoding the general benzoyl-CoA pathway of anaerobic aromatic metabolism. Rather, it was located in a cluster of genes coding for a novel aerobic benzoate oxidation pathway. In line with this finding, the same CoA ligase was induced during aerobic growth with benzoate. A deletion mutant not only was unable to grow anaerobically on benzoate or 2-aminobenzoate, but also aerobic growth on benzoate was affected. This suggests that benzoate induces a single benzoate-CoA ligase. The product of benzoate activation, benzoyl-CoA, then acts as inducer of separate anaerobic or aerobic pathways of benzoyl-CoA, depending on whether oxygen is lacking or present.

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