1999, Hille, Russ, Rétey, János, Bartlewski-Hof, Ulrike, Reichenbecher, Wolfram, Schink, Bernhard

In the past several years, the number of enzymes known to possess mononuclear molybdenum centers in their active sites has increased signi¢cantly, and well over 50 such enzymes have been identi¢ed that catalyze a variety of hydroxylation, oxygen atom transfer and other oxidation-reduction reactions. Many of these enzymes have been isolated from either obligate anaerobes or facultative anaerobes grown under anaerobic or microaerobic conditions, and are involved in a variety of anabolic, catabolic and energy-conserving metabolic pathways. There are now several enzymes having known crystal structure, and this new structural information has provided the basis for an increasingly detailed understanding of the mechanisms of action of these enzymes. In the present review, an overview of our present understanding of the mechanism of action of these enzymes will be presented, and those of a few selected enzymes will be considered in greater detail. Several alternative classi¢cation schemes have been suggested for these molybdenum-containing enzymes, but for the purposes of the present discussion they will be considered to fall into three families, based on the structures of their molybdenum centers in their oxidized Mo(VI) state (Fig. 1) [1]. These families include: (i) the molybdenum hydroxylases, a large and broadly dispersed family of enzymes that possess an MoOS unit and catalyze the hydroxylation of a broad range of aldehydes and aromatic heterocycles; (ii) the eukaryotic oxo transferases, a family that at present includes only sul¢te oxidase and the assimilatory nitrate reductases, enzymes which possess an MoO2 unit in their active sites and which catalyze oxygen atom transfer to or from a substrate; and ¢nally (iii) a diverse group of prokaryotic enzymes that catalyze either oxo atom transfer or other oxidation-reduction reactions. As will be seen below, however, some may catalyze
signi¢cantly more complex (and interesting) reactions. Enzymes of this last group have a common molybdenum center structure in which the metal is coordinated by a pair of dithiolene ligands contributed by an unusual pterin cofactor [2]. This cofactor is common to all the mononuclear molybdenum (and tungsten) enzymes, but whereas the ¢rst two families possess only a single equivalent bound to the metal, in members of the third family two equivalents of the cofactor coordinate to the metal. It should be emphasized that this ¢nal group is structurally more diverse than the ¢rst two, and can be subdivided into enzymes that possess an Mo-O(Ser), Mo-S(Cys) or Mo-Se(Se-Cys) group contributed by the polypeptide; in addition, some of these enzymes appear to possess an MoNS group rather than the more commonly encountered MoNO (see [1] for a review).

1999, Reichenbecher, Wolfram, Schink, Bernhard

Conversion of pyrogallol to phloroglucinol was studied with the molybdenum enzyme transhydroxylase of the strictly anaerobic fermenting bacterium Pelobacter acidigallici. Transhydroxylation experiments in H 2 18 O revealed that none of the hydroxyl groups of phloroglucinol was derived from water, confirming the concept that this enzyme transfers a hydroxyl group from the cosubstrate 1,2,3,5-tetrahydroxybenzene (tetrahydroxybenzene) to the acceptor pyrogallol, and simultaneously regenerates the cosubstrate. This concept requires a reaction which synthesizes the cofactor de novo to maintain a sufficiently high intracellular pool during growth. Some sulfoxides and aromatic N-oxides were found to act as hydroxyl donors to convert pyrogallol to tetrahydroxybenzene. Again, water was not the source of the added hydroxyl groups; the oxides reacted as cosubstrates in a transhydroxylation reaction rather than as true oxidants in a net hydroxylation reaction. No oxidizing agent was found that supported a formation of tetrahydroxybenzene via a net hydroxylation of pyrogallol. However, conversion of pyrogallol to phloroglucinol in the absence of tetrahydroxybenzene was achieved if little pyrogallol and a high amount of enzyme preparation was used which had been pre-exposed to air. Obviously, the enzyme was oxidized by air to form sufficient amounts of tetrahydroxybenzene from pyrogallol to start the reaction. A reaction mechanism is proposed which combines an oxidative hydroxylation with a reductive dehydroxylation via the molybdenum cofactor, and allows the transfer of a hydroxyl group between tetrahydroxybenzene and pyrogallol without involvement of water. With this, the transhydroxylase differs basically from all other hydroxylating molybdenum enzymes which all use water as hydroxyl source.

1999, Schöcke, Ludger, Schink, Bernhard

The pathway of fermentative benzoate degradation by the syntrophically fermenting bacterium Syntrophus gentianae was studied by measurement of enzyme activities in cell-free extracts. Benzoate was activated by a benzoate-CoA ligase reaction, forming AMP and pyrophosphate, which was subsequently cleaved by a membrane-bound proton-translocating pyrophosphatase. Glutaconyl-CoA (formed from hypothetical pimelyl-CoA and glutaryl-CoA intermediates) was decarboxylated to crotonyl-CoA by a sodium-ion-dependent membrane-bound glutaconyl-CoA decarboxylase, a biotin enzyme that could be inhibited by avidin. The overall energy budget of this fermentation could be balanced only if the dearomatizing reduction of benzoyl-CoA is assumed to produce cyclohexene carboxyl-CoA rather than cyclohexadiene carboxyl-CoA, although experimental evidence of this reaction is still insufficient. With this assumption, benzoate degradation by S. gentianae can be balanced to yield onethird to two-thirds of an ATP unit per benzoate degraded, in accordance with earlier measurements of whole-cell energetics.

1998, Gallus, Corinna, Schink, Bernhard

Anaerobic degradation of alpha-resorcylate (3,5-dihydroxybenzoate) was studied with the denitrifying strain AR-1, which was assigned to the described species Thauera aromatica. Alpha-Resorcylate degradation does not proceed via the benzoyl-CoA, the resorcinol, or the phloroglucinol pathway. Instead, a-resorcylate is converted to hydroxyhydroquinone (1,2,4-trihydroxybenzene) by dehydrogenative oxidation and decarboxylation. Nitrate, K3[Fe(CN)6], dichlorophenol indophenol, and the NAD+ analogue 3-acetylpyridine adeninedinucleotide were suitable electron acceptors for the oxidation reaction; NAD+ did not function as an electron acceptor. The oxidation reaction was strongly accelerated by the additional presence of the redox carrier phenazine methosulfate, which could also be used as sole electron acceptor. Oxidation of alpha-resorcylate with molecular oxygen in cell suspensions or in cell-free extracts of a-resorcylate- and nitrate-grown cells was also detected although this bacterium did not grow with a-resorcylate under an air atmosphere. Alpha-Resorcylate degradation to hydroxyhydroquinone proceeded in two steps. The a-resorcylate-oxidizing enzyme activity was membrane-associated and exhibited maximal activity at pH 8.0. The primary oxidation product was not hydroxyhydroquinone. Rather, formation of hydroxyhydroquinone by decarboxylation of the unknown intermediate in addition required the cytoplasmic fraction and needed lower pH values since hydroxyhydroquinone was not stable at alkaline pH.

1999, Heising, Silke, Richter, Lothar, Ludwig, Wolfgang, Schink, Bernhard

A green phototrophic bacterium was enriched with ferrous iron as sole electron donor and was isolated in defined coculture with a spirilloid chemoheterotrophic bacterium. The coculture oxidized ferrous iron to ferric iron with stoichiometric formation of cell mass from carbon dioxide. Sulfide, thiosulfate, or elemental sulfur was not used as electron donor in the light. Hydrogen or acetate in the presence of ferrous iron increased the cell yield of the phototrophic partner, and hydrogen could also be used as sole electron source. Complexed ferric iron was slowly reduced to ferrous iron in the dark, with hydrogen as electron source. Similar to Chlorobium limicola, the phototrophic bacterium contained bacteriochlorophyll c and chlorobactene as photosynthetic pigments, and also resembled representatives of this species morphologically. On the basis of 16S rRNA sequence comparisons, this organism clusters with Chlorobium, Prosthecochloris, and Pelodictyon species within the green sulfur bacteria phylum. Since the phototrophic partner in the coculture KoFox is only moderately related to the other members of the cluster, it is proposed as a new species, Chlorobium ferrooxidans. The chemoheterotrophic partner bacterium, strain KoFum, was isolated in pure culture with fumarate as sole substrate. The strain was identified as a member of the e-subclass of the Proteobacteria closely related to Geospirillum arsenophilum on the basis of physiological properties and 16S rRNA sequence comparison. The Geospirillum strain was present in the coculture only in low numbers. It fermented fumarate, aspartate, malate, or pyruvate to acetate, succinate, and carbon dioxide, and could reduce nitrate to dinitrogen gas. It was not involved in ferrous iron oxidation but possibly provided a thus far unidentified growth factor to the phototrophic partner.

1999, Müller, Jochen A., Galushko, Alexander S., Kappler, Andreas, Schink, Bernhard

The anaerobic bacterium Desulfobacterium cetonicum oxidized m-cresol completely with sulfate as electron acceptor. During growth, 3-hydroxybenzylsuccinate (identified by gas chromatography/mass spectroscopy and by comparison of high-performance liquid chromatography retention time and UV spectrum with a chemically synthesized reference compound) accumulated in the medium. This finding indicates that the methyl group of mcresol is activated by addition to fumarate as in the case of anaerobic toluene metabolism. In cell-free extracts of D. cetonicum, the formation of 3-hydroxybenzylsuccinate from m-cresol and fumarate was detected at an activity of 0.5 nmol min 1 (mg protein) 1. This reaction depended strictly on anoxic assay conditions. Treatment with air resulted in a complete loss of activity; however, some activity could be recovered after restoring anoxic conditions. The activity was slightly membrane-associated. 3-Hydroxybenzylsuccinate was degraded via CoA thioesterification and further oxidation to 3-hydroxybenzoyl-CoA as subsequent steps in the degradation pathway.

1998, Philipp, Bodo, Schink, Bernhard

The denitrifying bacterium Azoarcus anaerobius LuFRes1 grows anaerobically with resorcinol (1,3-dihydroxybenzene) as the sole source of carbon and energy. The anaerobic degradation of this compound was investigated in cell extracts. Resorcinol reductase, the key enzyme for resorcinol catabolism in fermenting bacteria, was not present in this organism. Instead, resorcinol was hydroxylated to hydroxyhydroquinone (HHQ; 1,2,4-trihydroxybenzene) with nitrate or K3Fe(CN)6 as the electron acceptor. HHQ was further oxidized with nitrate to 2-hydroxy-1,4-benzoquinone as identified by high-pressure liquid chromatography, UV/visible light spectroscopy, and mass spectroscopy. Average specific activities were 60 mU mg of protein21 for resorcinol hydroxylation and 150 mU mg of protein21 for HHQ dehydrogenation. Both activities were found nearly exclusively in the membrane fraction and were only barely detectable in extracts of cells grown with benzoate, indicating that both reactions were specific for resorcinol degradation. These findings suggest a new strategy of anaerobic degradation of aromatic compounds involving oxidative steps for destabilization of the aromatic ring, different from the reductive dearomatization mechanisms described so far.

1999, Schink, Bernhard, Benz, Marcus

Sediments develop by sedimentation of organic and inorganic residues of primary and secondary production as well as by inorganic precipitates, e.g., metal hydroxides, carbonates, silicates, and phosphates. The accumulation of this material at the bottom of freshwater lakes leads to an intensification of mainly microbial degradative activities which oxidise and transform the organic freight with concomitant reduction of oxygen and other electron acceptors. It is the activity of micro-organisms, especially of bacteria, which leads to the reduction of available electron acceptors, to an accumulation of reduced derivatives, and with that to changes of the redox potential in such sediments.
The basic processes involved in the degradation of organic matter by such microbial communities are known for a long time. As long as molecular oxygen is available it acts as the preferred electron acceptor, followed by nitrate, manganese(IV) oxide, iron(III) hydroxides, sulfate, and finally CO2 with the release of nitrite, ammonia, dinitrogen, manganese(II) and iron(II) carbonates, sulfides, and finally methane as products of microbial reductive activities (STUMM & MORGAN,1981). These preferences for the various acceptor systems are mainly determined by the redox potential and the availability of the redox systems under consideration, with the most positive ones at the beginning and the lower ones to the end, according to the scheme depicted in Table 18.1.

1999, Schneeberger, Anne, Frings, Jochen, Schink, Bernhard

Eubacterium acidaminophilum combines the oxidation of amino acids such as alanine or valine with the reduction of glycine to acetate in a two-substrate fermentation (Stickland reaction). In the absence of glycine, dense cell suspensions oxidized alanine or valine only to a small extent, with limited production of hydrogen and acetate. Experiments with 14C-labeled carbonate revealed that acetate was formed under these conditions by net reduction of CO2/HCO ; 14C-labeled formate was formed as an intermediate. E. acidaminophilum did not grow with hydrogen plus CO2 ; dense cell suspensions under H2/CO2 produced only very small amounts (60.5 mM) of acetate. There was no activity of carbon monoxide dehydrogenase, indicating that the glycine pathway was used for acetate synthesis. The results are explained on the basis of biochemical and energetic considerations.

1998, Cord-Ruwisch, Ralf, Lovley, Derek R., Schink, Bernhard

Pure cultures of Geobacter sulfurreducens and other Fe(III)-reducing bacteriaaccumulated hydrogen topartial pressures of 5 to 70 Pa with acetate, butyrate, benzoate, ethanol, lactate, or glucose as the electrondonor if electron release to an acceptor was limiting. G. sulfurreducens coupled acetateoxidation with electrontransfer to an anaerobic partner bacterium in the absence of ferric iron or other electron acceptors. Coculturesof G. sulfurreducens and Wolinella succinogenes with nitrate as the electron acceptor degraded acetate efficiently and grew with doubling times of 6 to 8 h. The hydrogen partial pressures in these acetate-degrading cocultures were considerably lower, in the range of 0.02 to 0.04 Pa. From these values and the concentrations of the other reactants, it was calculated that in this cooperation the free energy change available to G. sulfurreducens should be about 253 kJ per mol of acetate oxidized, assuming complete conversion of acetate to CO2 and H2. However, growth yields (18.5 g of dry mass per mol of acetate for the coculture, about 14 g for G. sulfurreducens) indicated considerably higher energy gains. These yield data, measurement of hydrogen production rates, and calculation of the diffusive hydrogen flux indicated that electron transfer in these cocultures may not proceed exclusively via interspecies hydrogen transfer but may also proceed through an alternative carrier system with higher redox potential, e.g., a c-type cytochrome that was found to be excreted by G. sulfurreducens into the culture fluid. Syntrophic acetate degradation was also possible with G. sulfurreducens and Desulfovibrio desulfuricans CSN but only with nitrate as electron acceptor. These cultures produced cell yields of 4.5 g of dry mass per mol of acetate, to which both partners contributed at about equal rates. These results demonstrate that some Fe(III)-reducing bacteria can oxidize organic compounds under Fe(III) limitation with the production of hydrogen, and they provide the first example of rapid acetate oxidation via interspecies electron transfer at moderate temperature.