3D reconstruction and flexibility of the hybrid engine Acetobacterium woodii F-ATP synthase.

The Na+-translocating F1FO ATP synthase from Acetobacterium woodii (AwF-ATP synthase) with a subunit stoichiometry of α3:ß3:γ:δ:ε:a:b2:(c2/3)9:c1 represents an evolutionary path between ATP-synthases and vacuolar ATPases, by containing a heteromeric rotor c-ring, composed of subunits c1, c2 and c3, and an extra loop (γ195-211) within the rotary γ subunit. Here, the recombinant AwF-ATP synthase was subjected to negative stain electron microscopy and single particle analysis. The reference free 2D class averages revealed high flexibility of the enzyme, wherein the F1 and FO domains distinctively bended to adopt multiple conformations. Moreover, both the F1 and FO domains tilted relative to each other to a maximum extent of 28° and 30°, respectively. The first 3D reconstruction of the AwF-ATP synthase was determined which accommodates well the modelled structure of the AwF-ATP synthase as well as the γ195-211-loop. Molecular simulations of the enzyme underlined the bending features and flexibility observed in the electron micrographs, and enabled assessment of the dynamics of the extra γ195-211-loop.

Microbiome for the Electrosynthesis of Chemicals from Carbon Dioxide.

The price for renewable electricity is rapidly decreasing, and the availability of such energy is expected to increase in the coming years. This is a welcomed outcome considering that mitigation of climate disruption due to the use of fossil carbon is reaching a critical stage. However, the economy will remain dependent on carbon-based chemicals and the problem of electricity storage persists. Therefore, the development of electrosynthetic processes that convert electricity and CO2 into chemicals and energy dense fuels, perhaps even food, would be desirable. Electrochemistry has been applied to the manufacture of many valuable products and at a large industrial scale, but it is difficult to produce multicarbon chemicals from CO2 by chemistry alone. Being that the biological world possesses expertise at the construction of C-C bonds, it is being examined in conjunction with electrochemistry to discover new ways of synthesizing chemicals from electricity and CO2. One approach is microbial electrosynthesis. This Account describes the development of a microbial electrosynthesis system by the authors. A biocathode consisting of a carbon-based electrode and a microbial community produced short chain fatty acids, primarily acetate. The device works by electrolysis of water, but microbes facilitate electron transfer from the cathode while reducing CO2 by the Wood-Ljungdahl pathway possessed by an Acetobacterium sp. While this acetogenic microorganism dominates the microbiome growing on the cathode surface, 13 total species of microbes overall were ecologically selected on the cathode and genomes for each have been assembled. The combined species may contribute to the stability of the microbiome, a common feature of naturally selected microbial communities. The microbial electrosynthesis system was demonstrated to operate continuously at a cathode for more than 2 years and could also be used with intermittent power, thus demonstrating the stability of the microbiome living at the cathode. In addition to the description of reactor design and startup procedures, the possible mechanisms of electron transfer are described in this Account. While mysteries remain to be solved, much evidence indicates that the microbiome may facilitate electron transfer by supplying catalyst(s) external to the bacterial cells and onto the cathode surface. This may be in the form of a hydrogen-producing catalyst that enhances hydrogen generation by an inert carbon-based electrode. Through the enrichment of the electrosynthetic microbiome along with several modifications in reactor design and operation, the productivity and efficiency were improved. In addition to the intrinsic value of the current products, coupling the process with a secondary stage might be used to produce more valuable products from the acetic acid stream such as lipids, biocrude oil, or higher value food supplements. Alternatively, additional work on the mechanism of electron transfer, reactor design/operation, and modification of the microbes through synthetic biology, particularly to enhance carbon efficiency into higher value chemicals, are the needed next steps to advance microbial electrosynthesis so that it may be used to transform renewable electrons and CO2 directly into products and help solve the problem of climate disruption.

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).

A caffeyl-coenzyme A synthetase initiates caffeate activation prior to caffeate reduction in the acetogenic bacterium Acetobacterium woodii.

The anaerobic acetogenic bacterium Acetobacterium woodii couples the reduction of caffeate with electrons derived from hydrogen to the synthesis of ATP by a chemiosmotic mechanism using sodium ions as coupling ions, but the enzymes involved remain to be established. Previously, the electron transfer flavoproteins EtfA and EtfB were found to be involved in caffeate respiration. By inverse PCR, we identified three genes upstream of etfA and etfB: carA, carB, and carC. carA encodes a potential coenzyme A (CoA) transferase, carB an acyl-CoA synthetase, and carC an acyl-CoA dehydrogenase. carA, -B, and -C are located together with etfA/carE and etfB/carD on one polycistronic message, indicating that CarA, CarB, and CarC are also part of the caffeate respiration pathway. The genetic data suggest an initial ATP-dependent activation of caffeate by CarB. To prove the proposed function of CarB, the protein was overproduced in Escherichia coli, and the recombinant protein was purified. Purified CarB activates caffeate to caffeyl-CoA in an ATP- and CoA-dependent reaction. The enzyme has broad pH and temperature optima and requires K(+) for activity. In addition to caffeate, it can use ρ-coumarate, ferulate, and cinnamate as substrates, with 50, 15, and 9%, respectively, of the activity obtained with caffeate. Expression of the car operon is induced not only by caffeate, ρ-coumarate, ferulate, and cinnamate but also by sinapate. There is no induction by ρ-hydroxybenzoate or syringate.

Biochemistry, evolution and physiological function of the Rnf complex, a novel ion-motive electron transport complex in prokaryotes.

Microbes have a fascinating repertoire of bioenergetic enzymes and a huge variety of electron transport chains to cope with very different environmental conditions, such as different oxygen concentrations, different electron acceptors, pH and salinity. However, all these electron transport chains cover the redox span from NADH + H(+) as the most negative donor to oxygen/H(2)O as the most positive acceptor or increments thereof. The redox range more negative than -320 mV has been largely ignored. Here, we have summarized the recent data that unraveled a novel ion-motive electron transport chain, the Rnf complex, that energetically couples the cellular ferredoxin to the pyridine nucleotide pool. The energetics of the complex and its biochemistry, as well as its evolution and cellular function in different microbes, is discussed.

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.