Biomedical subjects

Matthias Boll

Publications and source records attributed to Matthias Boll.

15 recordsLinked to original sources

PubMed2006

Cyclohexa-1,5-diene-1-carbonyl-coenzyme A (CoA) hydratases of Geobacter metallireducens and Syntrophus aciditrophicus: Evidence for a common benzoyl-CoA degradation pathway in facultative and strict anaerobes.

In the denitrifying bacterium Thauera aromatica, the central intermediate of anaerobic aromatic metabolism, benzoyl-coenzyme A (CoA), is dearomatized by the ATP-dependent benzoyl-CoA reductase to cyclohexa-1,5-diene-1-carbonyl-CoA (dienoyl-CoA). The dienoyl-CoA is further metabolized by a series of beta-oxidation-like reactions of the so-called benzoyl-CoA degradation pathway resulting in ring cleavage. Recently, evidence was obtained that obligately anaerobic bacteria that use aromatic growth substrates do not contain an ATP-dependent benzoyl-CoA reductase. In these bacteria, the reactions involved in dearomatization and cleavage of the aromatic ring have not been shown, so far. In this work, a characteristic enzymatic step of the benzoyl-CoA pathway in obligate anaerobes was demonstrated and characterized. Dienoyl-CoA hydratase activities were determined in extracts of Geobacter metallireducens (iron reducing), Syntrophus aciditrophicus (fermenting), and Desulfococcus multivorans (sulfate reducing) cells grown with benzoate. The benzoate-induced genes putatively coding for the dienoyl-CoA hydratases in the benzoate degraders G. metallireducens and S. aciditrophicus were heterologously expressed and characterized. Both gene products specifically catalyzed the reversible hydration of dienoyl-CoA to 6-hydroxycyclohexenoyl-CoA (Km, 80 and 35 microM; Vmax, 350 and 550 micromol min(-1) mg(-1), respectively). Neither enzyme had significant activity with cyclohex-1-ene-1-carbonyl-CoA or crotonyl-CoA. The results suggest that benzoyl-CoA degradation proceeds via dienoyl-CoA and 6-hydroxycyclohexanoyl-CoA in strictly anaerobic bacteria. The steps involved in dienoyl-CoA metabolism appear identical in all nonphotosynthetic anaerobic bacteria, although totally different benzene ring-dearomatizing enzymes are present in facultative and obligate anaerobes.

Acyl Coenzyme A↗

PubMed2005

Key enzymes in the anaerobic aromatic metabolism catalysing Birch-like reductions.

Several novel enzyme reactions have recently been discovered in the aromatic metabolism of anaerobic bacteria. Many of these reactions appear to be catalyzed by oxygen-sensitive enzymes by means of highly reactive radical intermediates. This contribution deals with two key reactions in this metabolism: the ATP-driven reductive dearomatisation of the benzene ring and the reductive removal of a phenolic hydroxyl group. The two reactions catalyzed by benzoyl-CoA reductase (BCR) and 4-hydroxybenzoyl-CoA reductase (4-HBCR) are both mechanistically difficult to achieve; both are considered to proceed in 'Birch-like' reductions involving single electron and proton transfer steps to the aromatic ring. The problem of both reactions is the extremely high redox barrier for the first electron transfer to the substrate (e.g., -1.9 V in case of a benzoyl-CoA (BCoA) analogue), which is solved in the two enzymes in different manners. Studying these enzymatic reactions provides insights into general principles of how oxygen-dependent reactions are replaced by alternative processes under anoxic conditions.

Adenosine Triphosphate↗

PubMed2005

Gene clusters involved in anaerobic benzoate degradation of Geobacter metallireducens.

The degradation of aromatic compounds follows different biochemical principles in aerobic and anaerobic microorganisms. While aerobes dearomatize and cleave the aromatic ring by oxygenases, facultative anaerobes utilize an ATP-dependent ring reductase for the dearomatization of the activated key intermediate benzoyl-coenzyme A (CoA). In this work, the aromatic metabolism was studied in the obligately anaerobic model organism Geobacter metallireducens. The gene coding for a putative carboxylic acid-CoA ligase was heterologously overexpressed and the gene product was characterized as a highly specific benzoate-CoA ligase catalysing the initial step of benzoate metabolism. However, no evidence for the presence of an ATP-dependent benzoyl-CoA reductase as observed in facultative anaerobes was obtained. In a proteomic approach benzoate-induced proteins were identified; the corresponding genes are organized in two clusters comprising 44 genes. Induction of representative genes during growth on benzoate was confirmed by reverse transcription polymerase chain reaction. The results obtained suggest that benzoate is activated to benzoyl-CoA, which is then reductively dearomatized to cyclohexa-1,5-diene-1-carbonyl-CoA, followed by beta-oxidation reactions to acetyl-CoA units, as in facultatively anaerobic bacteria. However, in G. metallireducens the process of reductive benzene ring dearomatization appears to be catalysed by a set of completely different protein components comprising putative molybdenum and selenocysteine containing enzymes.

Acyl Coenzyme A↗

PubMed2005

Dearomatizing benzene ring reductases.

The high resonance energy of the benzene ring is responsible for the relative resistance of aromatic compounds to biodegradation. Nevertheless, bacteria from nearly all physiological groups have been isolated which utilize aromatic growth substrates as the sole source of cell carbon and energy. The enzymatic dearomatization of the benzene nucleus by microorganisms is accomplished in two different manners. In aerobic bacteria the aromatic ring is dearomatized by oxidation, catalyzed by oxygenases. In contrast, anaerobic bacteria attack the aromatic ring by reductive steps. Key intermediates in the anaerobic aromatic metabolism are benzoyl-CoA and compounds with at least two meta-positioned hydroxyl groups (resorcinol, phloroglucinol and hydroxyhydroquinone). In facultative anaerobes, the reductive dearomatization of the key intermediate benzoyl-CoA requires a stoichiometric coupling to ATP hydrolysis, whereas reduction of the other intermediates is readily achieved with suitable electron donors. Obligately anaerobic bacteria appear to use a totally different enzymology for the reductive dearomatization of benzoyl-CoA including selenocysteine- and molybdenum- containing enzymes.

Adenosine Triphosphate↗

PubMed2005

Unusual reactions involved in anaerobic metabolism of phenolic compounds.

Aerobic bacteria use molecular oxygen as a common co-substrate for key enzymes of aromatic metabolism. In contrast, in anaerobes all oxygen-dependent reactions are replaced by a set of alternative enzymatic processes. The anaerobic degradation of phenol to a non-aromatic product involves enzymatic processes that are uniquely found in the aromatic metabolism of anaerobic bacteria: (i) ATP-dependent phenol carboxylation to 4-hydroxybenzoate via a phenylphosphate intermediate (biological Kolbe-Schmitt carboxylation); (ii) reductive dehydroxylation of 4-hydroxybenzoyl-CoA to benzoyl-CoA; and (iii) ATP-dependent reductive dearomatization of the key intermediate benzoyl-CoA in a 'Birch-like' reduction mechanism. This review summarizes the results of recent mechanistic studies of the enzymes involved in these three key reactions.

Bacteria, Anaerobic↗

PubMed2005

Novel bacterial molybdenum and tungsten enzymes: three-dimensional structure, spectroscopy, and reaction mechanism.

The molybdenum enzymes 4-hydroxybenzoyl-CoA reductase and pyrogallol-phloroglucinol transhydroxylase and the tungsten enzyme acetylene hydratase catalyze reductive dehydroxylation reactions, i.e., transhydroxylation between phenolic residues and the addition of water to a triple bond. Such activities are unusual for this class of enzymes, which carry either a mononuclear Mo or W center. Crystallization and subsequent structural analysis by high-resolution X-ray crystallography has helped to resolve the reaction centers of these enzymes to a degree that allows us to understand the interaction of the enzyme and the respective substrate(s) in detail, and to develop a concept for the respective reaction mechanism, at least in two cases.

Bacteria, Anaerobic↗

PubMed2004

Substrate binding and reduction of benzoyl-CoA reductase: evidence for nucleotide-dependent conformational changes.

Benzoyl-CoA reductase (BCR) from the denitrifying bacterium Thauera aromatica catalyzes the ATP driven two-electron reduction of the aromatic moiety of benzoyl-CoA (BCoA) to a nonaromatic cyclic diene (2 ATP/2 e(-)). The enzyme contains two similar but nonidentical ATP-binding sites of the acetate kinase/sugar kinase/Hsp70/actin family. To obtain further insights into the overall catalytic cycle of BCR, the binding affinities and stoichiometries of all substrates as well as their effects on reduction kinetics were studied by stopped-flow UV/vis spectroscopy, freeze-quench EPR spectroscopy, and equilibrium dialysis. BCR bound maximally two nucleotides and a single BCoA. The binding of a single nucleotide induced a molecular switch (BCR --> BCR) as evidenced as follows: (i) the reduction rate of BCR by sulfoxide radical anion was significantly decreased in the nucleotide-bound state, (ii) the binding of BCoA to the reduced enzyme strictly depended on bound nucleotides, and (iii) the nucleotide binding affinities increased up to 60-fold compared to the steady-state values. The "ATP-binding switch" is distinguished from the previously described "low-spin/high-spin switch" of a [4Fe-4S] cluster which strictly depends on ATP hydrolysis. The two nucleotide binding sites were occupied sequentially; binding constants of the two sites differed by a factor of 10-40. The kinetic data obtained suggest that the ATP-binding switch is a rather fast process (>100 s(-)(1)) with a switch equilibrium constant of 54 +/- 10. In contrast, the reverse switch back of the MgADP-bound enzyme (BCR --> BCR) is considered rate-limiting in the overall catalytic cycle of BCR (4 +/- 1 s(-)(1)). The binding of nucleotides did not affect the redox potential of the [4Fe-4S](+1/+2) clusters; the switch is rather considered to alter the kinetics of internal electron transfer. Implications for the overall catalytic cycle of benzoyl-CoA reductase are discussed and compared with other ATP-hydrolyzing enzymes.

Acyl Coenzyme A↗

PubMed2004

Crystallization of 4-hydroxybenzoyl-CoA reductase and the structure of its electron donor ferredoxin.

4-Hydroxybenzoyl-CoA reductase (4-HBCR) is a central enzyme in the metabolism of phenolic compounds in anaerobic bacteria. The enzyme catalyzes the reductive removal of the phenolic hydroxyl group from 4-hydroxybenzoyl-CoA, yielding benzoyl-CoA and water. 4-HBCR belongs to the xanthine oxidase (XO) family of molybdenum enzymes which occur as heterodimers, (alphabetagamma)(2). 4-HBCR contains two molybdopterins, four [2Fe-2S] and two [4Fe-4S] clusters and two FADs. A low-potential Allochromatium vinosum-type ferredoxin containing two [4Fe-4S] clusters serves as an in vivo electron donor for 4-HBCR. In this work, the oxygen-sensitive proteins 4-HBCR and the ferredoxin (TaFd) from Thauera aromatica were crystallized under anaerobic conditions. 4-HBCR crystallized with PEG 4000 and MPD as precipitant diffracted to about 1.6 A resolution and the crystals were highly suitable for X-ray structure analysis. Crystals of TaFd were obtained with (NH(4))(3)PO(4) as precipitant and revealed a solvent content of 77%, which is remarkably high for a small soluble protein. The structure of TaFd was solved at 2.9 A resolution by the molecular-replacement method using the highly related structure of the ferredoxin (CvFd) from A. vinosum as a model. Structural changes between the two ferredoxins around the [4Fe-4S] cluster can be correlated with their different redox potentials.

Crystallography, X-Ray↗

PubMed2004

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

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.

Binding Sites↗

PubMed2004

The bzd gene cluster, coding for anaerobic benzoate catabolism, in Azoarcus sp. strain CIB.

We report here that the bzd genes for anaerobic benzoate degradation in Azoarcus sp. strain CIB are organized as two transcriptional units, i.e., a benzoate-inducible catabolic operon, bzdNOPQMSTUVWXYZA, and a gene, bzdR, encoding a putative transcriptional regulator. The last gene of the catabolic operon, bzdA, has been expressed in Escherichia coli and encodes the benzoate-coenzyme A (CoA) ligase that catalyzes the first step in the benzoate degradation pathway. The BzdA enzyme is able to activate a wider range of aromatic compounds than that reported for other previously characterized benzoate-CoA ligases. The reduction of benzoyl-CoA to a nonaromatic cyclic intermediate is carried out by a benzoyl-CoA reductase (bzdNOPQ gene products) detected in Azoarcus sp. strain CIB extracts. The bzdW, bzdX, and bzdY gene products show significant similarity to the hydratase, dehydrogenase, and ring-cleavage hydrolase that act sequentially on the product of the benzoyl-CoA reductase in the benzoate catabolic pathway of Thauera aromatica. Benzoate-CoA ligase assays and transcriptional analyses based on lacZ-reporter fusions revealed that benzoate degradation in Azoarcus sp. strain CIB is subject to carbon catabolite repression by some organic acids, indicating the existence of a physiological control that connects the expression of the bzd genes to the metabolic status of the cell.

Anaerobiosis↗

PubMed2004

Selenocysteine-containing proteins in anaerobic benzoate metabolism of Desulfococcus multivorans.

The sulfate-reducing bacterium Desulfococcus multivorans uses various aromatic compounds as sources of cell carbon and energy. In this work, we studied the initial steps in the aromatic metabolism of this strictly anaerobic model organism. An ATP-dependent benzoate coenzyme A (CoA) ligase (AMP plus PPi forming) composed of a single 59-kDa subunit was purified from extracts of cells grown on benzoate. Specific activity was highest with benzoate and some benzoate derivatives, whereas aliphatic carboxylic acids were virtually unconverted. The N-terminal amino acid sequence showed high similarities with benzoate CoA ligases from Thauera aromatica and Azoarcus evansii. When cultivated on benzoate, cells strictly required selenium and molybdenum, whereas growth on nonaromatic compounds, such as cyclohexanecarboxylate or lactate, did not depend on the presence of the two trace elements. The growth rate on benzoate was half maximal with 1 nM selenite present in the growth medium. In molybdenum- and/or selenium-depleted cultures, growth on benzoate could be induced by addition of the missing trace elements. In extracts of cells grown on benzoate in the presence of [75Se]selenite, three radioactively labeled proteins with molecular masses of approximately 100, 30, and 27 kDa were detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. The 100- and 30-kDa selenoproteins were 5- to 10-fold induced in cells grown on benzoate compared to cells grown on lactate. These results suggest that the dearomatization process in D. multivorans is not catalyzed by the ATP-dependent Fe-S enzyme benzoyl-CoA reductase as in facultative anaerobes but rather involves unknown molybdenum- and selenocysteine-containing proteins.

Amino Acid Sequence↗

PubMed2003

2-Oxoglutarate:NADP(+) oxidoreductase in Azoarcus evansii: properties and function in electron transfer reactions in aromatic ring reduction.

The conversion of [(14)C]benzoyl-coenzyme A (CoA) to nonaromatic products in the denitrifying beta-proteobacterium Azoarcus evansii grown anaerobically on benzoate was investigated. With cell extracts and 2-oxoglutarate as the electron donor, benzoyl-CoA reduction occurred at a rate of 10 to 15 nmol min(-1) mg(-1). 2-Oxoglutarate could be replaced by dithionite (200% rate) and by NADPH ( approximately 10% rate); in contrast NADH did not serve as an electron donor. Anaerobic growth on aromatic compounds induced 2-oxoglutarate:acceptor oxidoreductase (KGOR), which specifically reduced NADP(+), and NADPH:acceptor oxidoreductase. KGOR was purified by a 76-fold enrichment. The enzyme had a molecular mass of 290 +/- 20 kDa and was composed of three subunits of 63 (gamma), 62 (alpha), and 37 (beta) kDa in a 1:1:1 ratio, suggesting an (alphabetagamma)(2) composition. The native enzyme contained Fe (24 mol/mol of enzyme), S (23 mol/mol), flavin adenine dinucleotide (FAD; 1.4 mol/mol), and thiamine diphosphate (0.95 mol/mol). KGOR from A. evansii was highly specific for 2-oxoglutarate as the electron donor and accepted both NADP(+) and oxidized viologens as electron acceptors; in contrast NAD(+) was not reduced. These results suggest that benzoyl-CoA reduction is coupled to the complete oxidation of the intermediate acetyl-CoA in the tricarboxylic acid cycle. Electrons generated by KGOR can be transferred to both oxidized ferredoxin and NADP(+), depending on the cellular needs. N-terminal amino acid sequence analysis revealed that the open reading frames for the three subunits of KGOR are similar to three adjacently located open reading frames in Bradyrhizobium japonicum. We suggest that these genes code for a very similar three-subunit KGOR, which may play a role in nitrogen fixation. The alpha-subunit is supposed to harbor one FAD molecule, two [4Fe-4S] clusters, and the NADPH binding site; the beta-subunit is supposed to harbor one thiamine diphosphate molecule and one further [4Fe-4S] cluster; and the gamma-subunit is supposed to harbor the CoA binding site. This is the first study of an NADP(+)-specific KGOR. A similar NADP(+)-specific pyruvate oxidoreductase, which contains all domains in one large subunit, has been reported for the mitochondrion of the protist Euglena gracilis and the apicomplexan Cryptosporidium parvum.

Acyl Coenzyme A↗

PubMed2002

A Birch-like mechanism in enzymatic benzoyl-CoA reduction: a kinetic study of substrate analogues combined with an ab initio model.

Benzoyl-CoA reductase from the anaerobic bacterium Thauera aromatica catalyzes the ATP-driven two-electron reduction of the aromatic moiety of benzoyl-CoA. A Birch mechanism involving alternate one-electron and one-proton transfer steps to the aromatic ring was previously proposed for benzoyl-CoA reductase. Due to the high redox barrier, the first electron transfer step yielding a radical anion is considered the rate-limiting step in this reaction. Focusing on the mechanism of substrate reduction, this work combines the kinetic analysis of a number of substrate analogues with a model based on the ab initio calculated electron density of the radical anion of benzoyl-CoA, a transition state model of the proposed Birch mechanism. Both K(m) and k(cat) of ortho-substituted benzoyl-CoA increased in parallel with the substituent's acceptor strength (F > Cl = H > OH > NH(2)). Among the isomers of monofluorobenzoyl-CoA, reduction rates decreased in the following order: ortho > meta > para; the K(m) values increased in the following order: meta > ortho > para. Five-ring heteroaromatic acid thiol esters were reduced in the following order: thiophene > furan > pyrrole; the 2-isomers are reduced much faster than the 3-isomers. Most of these results could be rationalized by the model. A Hammett plot indicated that the reaction mechanism is only slightly polar, suggesting the involvement of a partial protonation of the carbonyl oxygen of benzoyl-CoA and/or a simultaneous transfer of the first electron and proton. Surprisingly, benzoyl-CoA reductase exhibited a hydrogen kinetic isotope effect on k(cat) for pyridine-2-carbonyl-CoA (2.1) but only a negligible one for benzoyl-CoA (1.2), indicating that pyridine-2-carbonyl-CoA reduction proceeds according to a varied mechanism.

Acyl Coenzyme A↗

PubMed2002

Anaerobic oxidation of aromatic compounds and hydrocarbons.

Aromatic compounds and hydrocarbons have in common a great stability due to resonance energy and inertness of CbondH and CbondC bonds. It has been taken for granted that the metabolism of these compounds obligatorily depends on molecular oxygen. Oxygen is required first to introduce hydroxyl groups into the substrate and then to cleave the aromatic ring. However, newly discovered bacterial enzymes and reactions involved in oxidation of aromatic and hydrocarbon compounds to CO(2) in the complete absence of molecular oxygen have been discovered. Of special interest are two reactions: the reduction of the aromatic ring of benzoyl-coenzyme A and the addition of fumarate to hydrocarbons. These reactions transform aromatic rings and hydrocarbons into products that can be oxidized via more conventional beta-oxidation pathways.

Acyl Coenzyme A↗

PubMed2002

Properties of 2-oxoglutarate:ferredoxin oxidoreductase from Thauera aromatica and its role in enzymatic reduction of the aromatic ring.

Benzoyl coenzyme A (benzoyl-CoA) reductase is a key enzyme in the anaerobic metabolism of aromatic compounds catalyzing the ATP-driven reductive dearomatization of benzoyl-CoA. The enzyme from Thauera aromatica uses a reduced 2[4Fe-4S] ferredoxin as electron donor. In this work, we identified 2-oxoglutarate:ferredoxin oxidoreductase (KGOR) as the ferredoxin reducing enzyme. KGOR activity was increased 10- to 50-fold in T. aromatica cells grown under denitrifying conditions on an aromatic substrate compared to that of cells grown on nonaromatic substrates. The enzyme was purified from soluble extracts by a 60-fold enrichment with a specific activity of 4.8 micromol min(-1) mg(-1). The native enzyme had a molecular mass of 200 +/- 20 kDa (mean +/- standard deviation) and consisted of two subunits with molecular masses of 66 and 34 kDa, suggesting an (alphabeta)(2) composition. The UV/visible spectrum was characteristic for an iron-sulfur protein; the enzyme contained 8.3 +/- 0.5 mol of Fe, 7.2 +/- 0.5 mol of acid-labile sulfur, and 1.6 +/- 0.2 mol of thiamine diphosphate (TPP) per mol of protein. The high specificity for 2-oxoglutarate and the low K(m) for ferredoxin ( approximately 10 microM) indicated that both are the in vivo substrates of the enzyme. KGOR catalyzed the isotope exchange between (14)CO(2) and C(1) of 2-oxoglutarate, representing a typical reversible partial reaction of 2-oxoacid oxidoreductases. The two genes coding for the two subunits of KGOR were found adjacent to the gene cluster coding for enzymes and ferredoxin of the catabolic benzoyl-CoA pathway. Sequence comparisons with other 2-oxoacid oxidoreductases indicated that KGOR from T. aromatica belongs to the Halobacterium type of 2-oxoacid oxidoreductases, which lack a ferredoxin-like module which contains two additional [4Fe-4S](1+/2+) clusters/monomer. Using purified KGOR, ferredoxin, and benzoyl-CoA reductase, the 2-oxoglutarate-driven reduction of benzoyl-CoA was shown in vitro. This demonstrates that ferredoxin acts as an electron shuttle between the citric acid cycle and benzoyl-CoA reductase by coupling the oxidation of the end product of the benzoyl-CoA pathway, acetyl-CoA, to the reduction of the aromatic ring.

Amino Acid Sequence↗