Publications

• Structure of a xanthine oxidase-related 4-hydroxybenzoyl-CoA reductase with an additional [4Fe-4S] cluster and an inverted electron flow.

  Unciuleac M., Warkentin E., Page C.C., Boll M., Ermler U.

  The Mo-flavo-Fe/S-dependent heterohexameric protein complex 4-hydroxybenzoyl-CoA reductase (4-HBCR, dehydroxylating) is a central enzyme of the anaerobic degradation of phenolic compounds and belongs to the xanthine oxidase (XO) family of molybdenum enzymes. Its X-ray structure was established at ... >> More

  The Mo-flavo-Fe/S-dependent heterohexameric protein complex 4-hydroxybenzoyl-CoA reductase (4-HBCR, dehydroxylating) is a central enzyme of the anaerobic degradation of phenolic compounds and belongs to the xanthine oxidase (XO) family of molybdenum enzymes. Its X-ray structure was established at 1.6 A resolution. The most pronounced difference between 4-HBCR and other structurally characterized members of the XO family is the insertion of 40 amino acids within the beta subunit, which carries an additional [4Fe-4S] cluster at a distance of 16.5 A to the isoalloxazine ring of FAD. The architecture of 4-HBCR and concomitantly performed electron transfer rate calculations suggest an inverted electron transfer chain from the donor ferredoxin via the [4Fe-4S] cluster to the Mo over a distance of 55 A. The binding site of 4-hydroxybenzoyl-CoA is located in an 18 A long channel lined up by several aromatic side chains around the aromatic moiety, which are proposed to shield and stabilize the postulated radical intermediates during catalysis. << Less

  Structure 12:2249-2256(2004) [PubMed] [EuropePMC]

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

  Heider J., Boll M., Breese K., Breinig S., Ebenau-Jehle C., Feil U., Gad'on N., Laempe D., Leuthner B., Mohamed M.E., Schneider S., Burchhardt G., Fuchs G.

  Differential induction of enzymes involved in anaerobic metabolism of aromatic substrates was studied in the denitrifying bacterium Thauera aromatica. This metabolism is divided into (1) peripheral reactions transforming the aromatic growth substrates to the common intermediate benzoyl-CoA, (2) th ... >> More

  Differential induction of enzymes involved in anaerobic metabolism of aromatic substrates was studied in the denitrifying bacterium Thauera aromatica. This metabolism is divided into (1) peripheral reactions transforming the aromatic growth substrates to the common intermediate benzoyl-CoA, (2) the central benzoyl-CoA pathway comprising ring-reduction of benzoyl-CoA and subsequent beta-oxidation to 3-hydroxypimelyl-CoA, and (3) the pathway of beta-oxidation of 3-hydroxypimelyl-CoA to three acetyl-CoA and CO2. Regulation was studied by three methods. 1. Determination of protein patterns of cells grown on different substrates. This revealed several strongly substrate-induced polypeptides that were missing in cells grown on benzoate or other intermediates of the respective metabolic pathways. 2. Measurement of activities of known enzymes involved in this metabolism in cells grown on different substrates. The enzyme pattern found is consistent with the regulatory pattern deduced from simultaneous adaptation of cells to utilisation of other aromatic substrates. 3. Immunological detection of catabolic enzymes in cells grown on different substrates. Benzoate-CoA ligase and 4-hydroxybenzoate-CoA ligase were detected only in cells yielding the respective enzyme activity. However, presence of the subunits of benzoyl-CoA reductase and 4-hydroxybenzoyl-CoA reductase was also recorded in some cell batches lacking enzyme activity. This possibly indicates an additional level of regulation on protein level for these two reductases. << Less

  Arch Microbiol 170:120-131(1998) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• 4-hydroxybenzoyl-CoA reductase (dehydroxylating) from the denitrifying bacterium Thauera aromatica -- prosthetic groups, electron donor, and genes of a member of the molybdenum-flavin-iron-sulfur proteins.

  Breese K., Fuchs G.

  4-Hydroxybenzoyl-CoA reductase catalyzes an important reaction in the anaerobic metabolism of phenolic compounds, i.e. the reductive removal of an aromatic hydroxyl group. The prosthetic groups and the natural electron donor of the enzyme were investigated and the genes were cloned and sequenced. ... >> More

  4-Hydroxybenzoyl-CoA reductase catalyzes an important reaction in the anaerobic metabolism of phenolic compounds, i.e. the reductive removal of an aromatic hydroxyl group. The prosthetic groups and the natural electron donor of the enzyme were investigated and the genes were cloned and sequenced. The enzyme is a molybdenum-flavin-iron-sulfur protein of subunit composition of alpha2beta2gamma2. It contains approximately 1.3 flavin nucleotide, probably FAD, 1.9 Mo, 15 Fe, and 12.5 acid-labile sulfur. Sequence interpretation suggests that the native enzyme contains two [4Fe-4S] and four [2Fe-2S] clusters. A 9.8-kDa ferredoxin with two [4Fe-4S] clusters functions as the natural electron donor. The genes coding for the three subunits, hcrABC, show high similarities to other molybdenum-flavin-iron-sulfur proteins of the xanthine oxidase family, notably to the three putative 4-hydroxybenzoyl-CoA reductase genes in Rhodopseudomonas palustris. In addition, there are close similarities to three open reading frames (orf) in E. coli. A major difference is the presence of an additional domain in the beta-subunit (HcrB, 35 kDa) probably carrying an additional iron-sulfur cluster. The 82-kDa alpha-subunit (HcrA) contains a Mo-cofactor-binding site. The 17-kDa gamma-subunit (HcrC) harbors two [2Fe-2S] clusters. Upstream of the hcrCAB region, an ORF was found coding for a regulatory protein of the MarR family. Downstream of the hcrCAB region lies an ORF presumably coding for a hydrophobic permease. << Less

  Eur. J. Biochem. 251:916-923(1998) [PubMed] [EuropePMC]

• Redox centers of 4-hydroxybenzoyl-CoA reductase, a member of the xanthine oxidase family of molybdenum-containing enzymes.

  Boll M., Fuchs G., Meier C., Trautwein A., El Kasmi A., Ragsdale S.W., Buchanan G., Lowe D.J.

  4-Hydroxybenzoyl-CoA reductase (4-HBCR) is a key enzyme in the anaerobic metabolism of phenolic compounds. It catalyzes the reductive removal of the hydroxyl group from the aromatic ring yielding benzoyl-CoA and water. The subunit architecture, amino acid sequence, and the cofactor/metal content i ... >> More

  4-Hydroxybenzoyl-CoA reductase (4-HBCR) is a key enzyme in the anaerobic metabolism of phenolic compounds. It catalyzes the reductive removal of the hydroxyl group from the aromatic ring yielding benzoyl-CoA and water. The subunit architecture, amino acid sequence, and the cofactor/metal content indicate that it belongs to the xanthine oxidase (XO) family of molybdenum cofactor-containing enzymes. 4-HBCR is an unusual XO family member as it catalyzes the irreversible reduction of a CoA-thioester substrate. A radical mechanism has been proposed for the enzymatic removal of phenolic hydroxyl groups. In this work we studied the spectroscopic and electrochemical properties of 4-HBCR by EPR and Mössbauer spectroscopy and identified the pterin cofactor as molybdopterin mononucleotide. In addition to two different [2Fe-2S] clusters, one FAD and one molybdenum species per monomer, we also identified a [4Fe-4S] cluster/monomer, which is unique among members of the XO family. The reduced [4Fe-4S] cluster interacted magnetically with the Mo(V) species, suggesting that the centers are in close proximity, (<15 A apart). Additionally, reduction of the [4Fe-4S] cluster resulted in a loss of the EPR signals of the [2Fe-2S] clusters probably because of magnetic interactions between the Fe-S clusters as evidenced in power saturation studies. The Mo(V) EPR signals of 4-HBCR were typical for XO family members. Under steady-state conditions of substrate reduction, in the presence of excess dithionite, the [4Fe-4S] clusters were in the fully oxidized state while the [2Fe-2S] clusters remained reduced. The redox potentials of the redox cofactors were determined to be: [2Fe-2S](+1/+2) I, -205 mV; [2Fe-2S] (+1/+2) II, -255 mV; FAD/FADH( small middle dot)/FADH, -250 mV/-470 mV; [4Fe-4S](+1/+2), -465 mV and Mo(VI)/(V)/(VI), -380 mV/-500 mV. A catalytic cycle is proposed that takes into account the common properties of molybdenum cofactor enzymes and the special one-electron chemistry of dehydroxylation of phenolic compounds. << Less

  J. Biol. Chem. 276:47853-47862(2001) [PubMed] [EuropePMC]

• Hydroxybenzoyl-CoA reductase: coupling kinetics and electrochemistry to derive enzyme mechanisms.

  el Kasmi A., Brachmann R., Fuchs G., Ragsdale S.W.

  Hydroxybenzoyl-CoA reductase (HBCR) is an iron-sulfur protein that is involved in the metabolism of aromatic compounds. It catalyzes the two-electron reduction of hydroxybenzoyl-CoA to benzoyl-CoA. In the work described here, kinetic schemes were derived for HBCR and for several classes of redox e ... >> More

  Hydroxybenzoyl-CoA reductase (HBCR) is an iron-sulfur protein that is involved in the metabolism of aromatic compounds. It catalyzes the two-electron reduction of hydroxybenzoyl-CoA to benzoyl-CoA. In the work described here, kinetic schemes were derived for HBCR and for several classes of redox enzymes and redox-activated enzymes. Introduction of the Nernst equation into the rate laws led to the development of novel relationships between the ambient redox potential, the midpoint potential of the enzyme active site, and the kinetic parameter, V/K. By coupling electrochemistry and steady-state kinetics, mechanistic information could be obtained that could not be determined by either method alone. For HBCR, the relationship between the kinetic parameter V/K and the ambient electrochemical potential of the assay mixture was found to be: apparent V/Km = Vmax/(Km(1 + exp[nF/RT(E - E(o)e)])), where n is the number of electrons involved in the redox process, F is the Faraday constant, R is the gas constant, T is the temperature in K, E is the applied potential, and E(o)e is the redox potential of a redox-active catalytic site on the enzyme. Coupling kinetics with electrochemistry yielded the E(o)e (-350 mV vs NHE) for HBCR and maximum values under optimal redox conditions for kcat and kcat/Km (9 s-1 and 1.8 x 10(5) M-1 s-1, respectively). In addition, theory was developed that could distinguish a single two-electron transfer mechanism from one involving two successive one-electron transfers. HBCR was found to be in the latter class. Interestingly, the derived mechanism for HBCR is similar to that of the Birch reduction, the classical organic chemical reaction for reductive dehydroxylation of phenolic compounds. The methodology described here represents a novel approach that should help elucidate the mechanisms of other oxidoreductase and redox-activated enzymes. << Less

  Biochemistry 34:11668-11677(1995) [PubMed] [EuropePMC]

• Reductive dehydroxylation of 4-hydroxybenzoyl-CoA to benzoyl-CoA in a denitrifying, phenol-degrading Pseudomonas species.

  Glockler R., Tschech A., Fuchs G.

  The initial reactions in anaerobic degradation of phenol to CO2 have been studied in vitro with a denitrifying Pseudomonas strain grown with phenol and nitrate in the absence of molecular oxygen. Phenol has been proposed to be carboxylated to 4-hydroxybenzoate [(1987) Arch. Microbiol. 148, 213-217 ... >> More

  The initial reactions in anaerobic degradation of phenol to CO2 have been studied in vitro with a denitrifying Pseudomonas strain grown with phenol and nitrate in the absence of molecular oxygen. Phenol has been proposed to be carboxylated to 4-hydroxybenzoate [(1987) Arch. Microbiol. 148, 213-217]. 4-Hydroxybenzoate was activated to 4-hydroxybenzoyl-CoA by a coenzyme A ligase. Cell extracts also catalyzed the reductive dehydroxylation of 4-hydroxybenzoyl-CoA to benzoyl-CoA with reduced benzyl viologen as electron donor. This enzyme, benzoyl-CoA:(acceptor) 4-oxidoreductase (hydroxylating) (EC 1.3.99.-), has not been reported before. The data suggest that phenol and 4-hydroxybenzoate are anaerobically metabolized by this strain via benzoyl-CoA. << Less

  FEBS Lett 251:237-240(1989) [PubMed] [EuropePMC]

• Anaerobic metabolism of aromatic compounds.

  Heider J., Fuchs G.

  Aromatic compounds comprise a wide variety of low-molecular-mass natural compounds (amino acids, quinones, flavonoids, etc.) and biopolymers (lignin, melanin). They are almost exclusively degraded by microorganisms. Aerobic aromatic metabolism is characterised by the extensive use of molecular oxy ... >> More

  Aromatic compounds comprise a wide variety of low-molecular-mass natural compounds (amino acids, quinones, flavonoids, etc.) and biopolymers (lignin, melanin). They are almost exclusively degraded by microorganisms. Aerobic aromatic metabolism is characterised by the extensive use of molecular oxygen. Monoxygenases and dioxygenases are essential for the hydroxylation and cleavage of aromatic ring structures. Accordingly, the characteristic central intermediates of the aerobic pathways (e.g. catechol) are readily attacked oxidatively. Anaerobic aromatic catabolism requires, of necessity, a quite different strategy. The basic features of this metabolism have emerged from studies on bacteria that degrade soluble aromatic substrates to CO2 in the complete absence of molecular oxygen. Essential to anaerobic aromatic metabolism is the replacement of all the oxygen-dependent steps by an alternative set of novel reactions and the formation of different central intermediates (e.g. benzoyl-CoA) for breaking the aromaticity and cleaving the ring; notably, in anaerobic pathways, the aromatic ring is reduced rather than oxidised. The two-electron reduction of benzoyl-CoA to a cyclic diene requires the cleavage of two molecules of ATP to ADP and P1 and is catalysed by benzoyl-CoA reductase. After nitrogenase, this is the second enzyme known which overcomes the high activation energy required for reduction of a chemically stable bond by coupling electron transfer to the hydrolysis of ATP. The alicyclic product cyclohex-1,5-diene-1-carboxyl-CoA is oxidised to acetyl-CoA via a modified beta-oxidation pathway; the ring structure is opened hydrolytically. Some phenolic compounds are anaerobically transformed to resorcinol (1,3-dihydroxybenzene) or phloroglucinol (1,3,5-trihydroxybenzene). These intermediates are also first reduced and then as alicyclic products oxidised to acetyl-CoA. This review gives an outline of the anaerobic pathways which allow bacteria to utilize aromatics even in the absence of oxygen. We focus on previously unknown reactions and on the enzymes characteristic for such novel metabolism. << Less

  Eur J Biochem 243:577-596(1997) [PubMed] [EuropePMC]