Flavodoxin hydroquinone provides electrons for the ATP-dependent reactivation of protein-bound corrinoid cofactors.

Corrinoid-dependent enzyme systems rely on the super-reduced state of the protein-bound corrinoid cofactor to be functional, for example, in methyl transfer reactions. Due to the low redox potential of the [CoII ]/[CoI ] couple, autoxidation of the corrinoid cofactor occurs and leads to the formation of the inactive [CoII ]-state. For the reactivation, which is an energy-demanding process, electrons have to be transferred from a physiological donor to the corrinoid cofactor by the help of a reductive activator protein. In this study, we identified reduced flavodoxin as electron donor for the ATP-dependent reduction of protein-bound corrinoid cofactors of bacterial O-demethylase enzyme systems. Reduced flavodoxin was generated enzymatically using pyruvate:ferredoxin/flavodoxin oxidoreductase rather than hydrogenase. Two of the four flavodoxins identified in Acetobacterium dehalogenans and Desulfitobacterium hafniense DCB-2 were functional in supplying electrons for corrinoid reduction. They exhibited a midpoint potential of about -400 mV (ESHE , pH 7.5) for the semiquinone/hydroquinone transition. Reduced flavodoxin could be replaced by reduced clostridial ferredoxin. It was shown that the low-potential electrons of reduced flavodoxin are first transferred to the iron-sulfur cluster of the reductive activator and finally to the protein-bound corrinoid cofactor. This study further highlights the importance of reduced flavodoxin, which allows maintaining a variety of enzymatic reaction cycles by delivering low-potential electrons.

Redox potential changes during ATP-dependent corrinoid reduction determined by redox titrations with europium(II)-DTPA.

Corrinoids are essential cofactors of enzymes involved in the C1 metabolism of anaerobes. The active, super-reduced [CoI ] state of the corrinoid cofactor is highly sensitive to autoxidation. In O-demethylases, the oxidation to inactive [CoII ] is reversed by an ATP-dependent electron transfer catalyzed by the activating enzyme (AE). The redox potential changes of the corrinoid cofactor, which occur during this reaction, were studied by potentiometric titration coupled to UV/visible spectroscopy. By applying europium(II)-diethylenetriaminepentaacetic acid (DTPA) as a reductant, we were able to determine the midpoint potential of the [CoII ]/[CoI ] couple of the protein-bound corrinoid cofactor in the absence and presence of AE and/or ATP. The data revealed that the transfer of electrons from a physiological donor to the corrinoid as the electron-accepting site is achieved by increasing the potential of the corrinoid cofactor from -530 ± 15 mV to -250 ± 10 mV (ESHE , pH 7.5). The first 50 to 100 mV of the shift of the redox potential seem to be caused by the interaction of nucleotide-bound AE with the corrinoid protein or its cofactor. The remaining 150-200 mV had to be overcome by the chemical energy of ATP hydrolysis. The experiments revealed that Eu(II)-DTPA, which was already known as a powerful reducing agent, is a suitable electron donor for titration experiments of low-potential redox centers. Furthermore, the results of this study will contribute to the understanding of thermodynamically unfavorable electron transfer processes driven by the power of ATP hydrolysis.

Community dynamics in a nitrate-reducing microbial consortium cultivated with p-alkylated vs. non-p-alkylated aromatic compounds.

In this study, we established the nitrate-reducing, aromatic compound-degrading enrichment culture pMB18. Its community structure was controlled by the aromatic substrate applied. In the presence of a p-alkylated substrate, microorganisms related to Sulfuritalea, Ignavibacterium and Comamonadaceae were abundant. Non-p-alkylated structural analogues promoted the enrichment of Azoarcus, which was probably favored by the excretion of nitrite. The analysis of the bamA gene, which is a functional marker for anaerobic aromatic compound degradation, as well as a differential abundance analysis suggested the involvement of Sulfuritalea and Comamonadaceae in the degradation of p-alkylated substrates. Members of the genus Azoarcus were assumed to be the key players for the degradation of the non-p-alkylated substrates. A gene cluster encoding a putative 4-methylbenzoyl-CoA reductase, which is supposed to be specific for the dearomatization of p-alkylated benzoyl-CoA intermediates, was detected in culture pMB18 dominated by Sulfuritalea, Ignavibacterium and Comamonadaceae, but not in an Azoarcus-dominated culture. This study allowed insight into a microbial community, whose composition was guided by the aromatic substrate applied.

Anaerobic aromatic compound degradation in Sulfuritalea hydrogenivorans sk43H.

Sulfuritalea hydrogenivorans sk43H is well recognized as a chemolithoautotrophic microorganism that oxidizes thiosulfate, sulfur or hydrogen. In this study, pathways for aromatic compound degradation were identified in the respective genome and proved for functionality by cultivation. S. hydrogenivorans sk43H harbors gene clusters encoding pathways for the anaerobic degradation of benzoate and phenylacetate via benzoyl-CoA as well as a partial pathway for anaerobic cinnamate degradation. Aerobic hybrid pathways were identified for the degradation of benzoate and 2-aminobenzoate. An aerobic pathway involving mono- and dioxygenases was found for 4-hydroxybenzoate. The organization of the gene clusters for anaerobic aromatic compound degradation in S. hydrogenivorans sk43H was found to be similar to that of the corresponding gene clusters in 'Aromatoleum aromaticum' strain EbN1. Cultivation experiments revealed that S. hydrogenivorans sk43H degrades benzoate, 4-hydroxybenzoate, phenylacetate and 4-hydroxyphenylacetate under nitrate-reducing conditions. The results imply a so far overlooked role of this microorganism in anaerobic aromatic compound degradation. Due to the frequent detection of Sulfuritalea-related microorganisms at hydrocarbon-contaminated sites, an involvement of this genus in the degradation of aromatic pollutants should be considered.

Microbial community of a gasworks aquifer and identification of nitrate-reducing Azoarcus and Georgfuchsia as key players in BTEX degradation.

We analyzed a coal tar polluted aquifer of a former gasworks site in Thuringia (Germany) for the presence and function of aromatic compound-degrading bacteria (ACDB) by 16S rRNA Illumina sequencing, bamA clone library sequencing and cultivation attempts. The relative abundance of ACDB was highest close to the source of contamination. Up to 44% of total 16S rRNA sequences were affiliated to ACDB including genera such as Azoarcus, Georgfuchsia, Rhodoferax, Sulfuritalea (all Betaproteobacteria) and Pelotomaculum (Firmicutes). Sequencing of bamA, a functional gene marker for the anaerobic benzoyl-CoA pathway, allowed further insights into electron-accepting processes in the aquifer: bamA sequences of mainly nitrate-reducing Betaproteobacteria were abundant in all groundwater samples, whereas an additional sulfate-reducing and/or fermenting microbial community (Deltaproteobacteria, Firmicutes) was restricted to a highly contaminated, sulfate-depleted groundwater sampling well. By conducting growth experiments with groundwater as inoculum and nitrate as electron acceptor, organisms related to Azoarcus spp. were identified as key players in the degradation of toluene and ethylbenzene. An organism highly related to Georgfuchsia toluolica G5G6 was enriched with p-xylene, a particularly recalcitrant compound. The anaerobic degradation of p-xylene requires a metabolic trait that was not described for members of the genus Georgfuchsia before. In line with this, we were able to identify a putative 4-methylbenzoyl-CoA reductase gene cluster in the respective enrichment culture, which is possibly involved in the anaerobic degradation of p-xylene.

Enrichment of Desulfitobacterium spp. from forest and grassland soil using the O-demethylation of phenyl methyl ethers as a growth-selective process.

The O-demethylation of phenyl methyl ethers under anaerobic conditions is a metabolic feature of acetogens and Desulfitobacterium spp. Desulfitobacteria as well as most acetogens are Gram-positive bacteria with a low GC content and belong to the phylum Firmicutes. The consumption of the phenyl methyl ether syringate was studied in enrichment cultures originating from five different topsoils. Desulfitobacterium spp. were detected in all topsoils via quantitative PCR. Desulfitobacteria could be enriched using the O-demethylation of syringate as a growth-selective process. The enrichment was significantly favoured by an external electron acceptor such as 3-chloro-4-hydroxyphenylacetate or thiosulfate. Upon cultivation in the presence of syringate and thiosulfate, which naturally occur in soil, a maximum number of 16S rRNA gene copies of Desulfitobacterium spp. was reached within the first three subcultivation steps and accounted for 3-10% of the total microbial community depending on the soil type. Afterwards, a loss of Desulfitobacterium gene copies was observed. Community analyses revealed that Proteobacteria, Acidobacteria, Actinobacteria and Bacteroidetes were the main phyla in the initial soil samples. Upon addition of syringate and thiosulfate as growth substrates, these phyla were rapidly outcompeted by Firmicutes, which were under-represented in soil. The main Firmicutes genera identified were Alkalibaculum, Clostridium, Sporobacterium, Sporomusa and Tissierella, which might be responsible for outcompeting the desulfitobacteria. Most of these organisms belong to the acetogens, which have previously been described to demethylate phenyl methyl ethers. The shift of the native community structure to almost exclusively Firmicutes supports the participation of members of this phylum in environmental demethylation processes.

Conversion of phenyl methyl ethers by Desulfitobacterium spp. and screening for the genes involved.

Microbial growth coupled to O-demethylation of phenyl methyl ethers, which are lignin decomposition products, was described for acetogenic bacteria and recently also for two species belonging to the nonacetogenic genus Desulfitobacterium. To elucidate the potential role of desulfitobacteria in the O-demethylation of phenyl methyl ethers in the environment, we cultivated Desulfitobacterium chlororespirans, D. dehalogenans, D. metallireducens, and different strains of D. hafniense with phenyl methyl ethers as sole electron donors. With the exception of D. metallireducens, all species and strains tested were able to demethylate at least three of the four phenyl methyl ethers applied with fumarate, nitrate, or thiosulfate as electron acceptor. Furthermore, a high number of operons putatively encoding demethylase systems were identified in the genomes of Desulfitobacterium spp., although discrimination between O-, S-, N- and, Cl-demethylases was not possible. These findings provide evidence for the importance of the methylotrophic metabolism for desulfitobacteria and point to their involvement in the O-demethylation of phenyl methyl ethers in the environment.

Kinetic regulation of a corrinoid-reducing metallo-ATPase by its substrates.

Corrinoid cofactors play a crucial role as methyl group carriers in the C1 metabolism of anaerobes, e.g. in the cleavage of phenyl methyl ethers by O-demethylases. For the methylation, the protein-bound corrinoid has to be in the super-reduced [Co(I) ]-state, which is highly sensitive to autoxidation. The reduction of inadvertently oxidized corrinoids ([Co(II) ]-state) is catalysed in an ATP-dependent reaction by RACE proteins, the reductive activators of corrinoid-dependent enzymes. In this study, a reductive activator of O-demethylase corrinoid proteins was characterized with respect to its ATPase and corrinoid reduction activity. The reduction of the corrinoid cofactor was dependent on the presence of potassium or ammonium ions. In the absence of the corrinoid protein, a basal slow ATP hydrolysis was observed which was obviously not coupled to corrinoid reduction. ATP hydrolysis was significantly stimulated by the corrinoid protein in the [Co(II) ]-state of the corrinoid cofactor. The stoichiometry of ATP hydrolysed per mol corrinoid reduced was near 1:1. Site-directed mutagenesis was applied to study the impact of a highly conserved region possibly involved in nucleotide binding of RACE proteins, indicating that an aspartate and a glycine residue may play an essential role for the function of the enzyme.

Corrinoid activation by a RACE protein: studies on the interaction of the proteins involved.

The O-demethylases of anaerobes are corrinoid-dependent, ether-cleaving methyltransferase enzyme systems consisting of four components. The interaction of the O-demethylase components of the acetogenic bacterium Acetobacterium dehalogenans was studied by protein mobility on native PAGE, far-Western blot analysis and yeast two-hybrid screen. Using native PAGE and far-Western blot, the interaction of the activating enzyme (AE) with its substrate, the corrinoid protein (CP), could be observed. The interaction occurred with four different CPs of A. dehalogenans and a CP from Desulfitobacterium hafniense DCB-2, all involved in ether cleavage. In the corrinoid reduction assay, the AE reduced all CPs tested. This result indicates a broad substrate specificity of the AE of A. dehalogenans. In addition, an interaction of the A. dehalogenans CP of the vanillate-O-demethylase with the two methyltransferases of the same enzyme system was observed. The interaction of the ether-cleaving methyltransferase with the CP appeared to be significantly less pronounced than that reported for the homologous methanol and methylamine methyltransferase systems of methanogenic archaea.

Characterization of an O-demethylase of Desulfitobacterium hafniense DCB-2.

Besides acetogenic bacteria, only Desulfitobacterium has been described to utilize and cleave phenyl methyl ethers under anoxic conditions; however, no ether-cleaving O-demethylases from the latter organisms have been identified and investigated so far. In this study, genes of an operon encoding O-demethylase components of Desulfitobacterium hafniense strain DCB-2 were cloned and heterologously expressed in Escherichia coli. Methyltransferases I and II were characterized. Methyltransferase I mediated the ether cleavage and the transfer of the methyl group to the superreduced corrinoid of a corrinoid protein. Desulfitobacterium methyltransferase I had 66% identity (80% similarity) to that of the vanillate-demethylating methyltransferase I (OdmB) of Acetobacterium dehalogenans. The substrate spectrum was also similar to that of the latter enzyme; however, Desulfitobacterium methyltransferase I showed a higher level of activity for guaiacol and used methyl chloride as a substrate. Methyltransferase II catalyzed the transfer of the methyl group from the methylated corrinoid protein to tetrahydrofolate. It also showed a high identity (∼70%) to methyltransferases II of A. dehalogenans. The corrinoid protein was produced in E. coli as cofactor-free apoprotein that could be reconstituted with hydroxocobalamin or methylcobalamin to function in the methyltransferase I and II assays. Six COG3894 proteins, which were assumed to function as activating enzymes mediating the reduction of the corrinoid protein after an inadvertent oxidation of the corrinoid cofactor, were studied with respect to their abilities to reduce the recombinant reconstituted corrinoid protein. Of these six proteins, only one was found to catalyze the reduction of the corrinoid protein.

The ether-cleaving methyltransferase of the strict anaerobe Acetobacterium dehalogenans: analysis of the zinc-binding site.

The anaerobic phenyl methyl ether cleavage in acetogenic bacteria is mediated by multicomponent enzyme systems designated O-demethylases. Depending on the growth substrate, different O-demethylases are induced in Acetobacterium dehalogenans. A vanillate- and a veratrol-O-demethylase of this organism have been described earlier. The methyltransferase I (MT I), a component of this enzyme system, catalyzes the ether cleavage and the transfer of the methyl group to a super-reduced corrinoid bound to a protein. The MT I of the vanillate- and veratrol-O-demethylase (MT I(van) and MT I(ver)) were found to be zinc-containing enzymes. By site-directed mutagenesis, putative zinc ligands were identified, from which the following unique zinc-binding motifs were derived: E-X(14)-E-X(20)-H for MT I(van) and D-X(27)-C-X(39)-C for MT I(ver).

Ether cleaving methyltransferases of the strict anaerobe Acetobacterium dehalogenans: controlling the substrate spectrum by genetic engineering of the N-terminus.

The anaerobic cleavage of ether bonds of methoxylated substrates such as vanillate or veratrol in acetogenic bacteria is mediated by multi-component enzyme systems, the O-demethylases. Acetobacterium dehalogenans harbours different inducible O-demethylases with various substrate spectra. Two of these enzyme systems, the vanillate- and the veratrol-O-demethylases, have been characterized so far. One component of this enzyme system, the methyltransferase I (MT I), catalyses the cleavage of the substrate ether bond and the subsequent transfer of the methyl group to a corrinoid protein. For the C-termini of the methyltransferases I of the vanillate- and the veratrol-O-demethylases, a TIM barrel structure of the enzymes was predicted, whereas the N-termini are not part of this conserved structure. The deletion of the N-terminal regions led to a significant increase of activity (up to 20-fold) and an extended substrate spectrum of the mutants, which also comprised non-aromatic compounds such as the thioether methionine and diethylether. The exchange of the N-termini of the two methyltransferases I resulted in chimeric enzymes whose substrate specificities were those of the enzymes from which the N-termini were derived. This demonstrated the crucial role of the N-termini for the substrate specificity of the methyltransferases.

The ether-cleaving methyltransferase system of the strict anaerobe Acetobacterium dehalogenans: analysis and expression of the encoding genes.

Anaerobic O-demethylases are inducible multicomponent enzymes which mediate the cleavage of the ether bond of phenyl methyl ethers and the transfer of the methyl group to tetrahydrofolate. The genes of all components (methyltransferases I and II, CP, and activating enzyme [AE]) of the vanillate- and veratrol-O-demethylases of Acetobacterium dehalogenans were sequenced and analyzed. In A. dehalogenans, the genes for methyltransferase I, CP, and methyltransferase II of both O-demethylases are clustered. The single-copy gene for AE is not included in the O-demethylase gene clusters. It was found that AE grouped with COG3894 proteins, the function of which was unknown so far. Genes encoding COG3894 proteins with 20 to 41% amino acid sequence identity with AE are present in numerous genomes of anaerobic microorganisms. Inspection of the domain structure and genetic context of these orthologs predicts that these are also reductive activases for corrinoid enzymes (RACEs), such as carbon monoxide dehydrogenase/acetyl coenzyme A synthases or anaerobic methyltransferases. The genes encoding the O-demethylase components were heterologously expressed with a C-terminal Strep-tag in Escherichia coli, and the recombinant proteins methyltransferase I, CP, and AE were characterized. Gel shift experiments showed that the AE comigrated with the CP. The formation of other protein complexes with the O-demethylase components was not observed under the conditions used. The results point to a strong interaction of the AE with the CP. This is the first report on the functional heterologous expression of acetogenic phenyl methyl ether-cleaving O-demethylases.

Veratrol-O-demethylase of Acetobacterium dehalogenans: ATP-dependent reduction of the corrinoid protein.

The anaerobic veratrol O-demethylase mediates the transfer of the methyl group of the phenyl methyl ether veratrol to tetrahydrofolate. The primary methyl group acceptor is the cobalt of a corrinoid protein, which has to be in the +1 oxidation state to bind the methyl group. Due to the negative redox potential of the cob(II)/cob(I)alamin couple, autoxidation of the cobalt may accidentally occur. In this study, the reduction of the corrinoid to the superreduced [Co(I)] state was investigated. The ATP-dependent reduction of the corrinoid protein of the veratrol O-demethylase was shown to be dependent on titanium(III) citrate as electron donor and on an activating enzyme. In the presence of ATP, activating enzyme, and Ti(III), the redox potential versus the standard hydrogen electrode (E (SHE)) of the cob(II)alamin/cob(I)alamin couple in the corrinoid protein was determined to be -290 mV (pH 7.5), whereas E (SHE) at pH 7.5 was lower than -450 mV in the absence of either activating enzyme or ATP. ADP, AMP, or GTP could not replace ATP in the activation reaction. The ATP analogue adenosine-5'-(beta,gamma-imido)triphosphate (AMP-PNP, 2-4 mM) completely inhibited the corrinoid reduction in the presence of ATP (2 mM).