Enzymes of anaerobic ethylbenzene and p-ethylphenol catabolism in 'Aromatoleum aromaticum': differentiation and differential induction.

The denitrifying bacterium 'Aromatoleum aromaticum' strain EbN1 is one of the best characterized bacteria regarding anaerobic ethylbenzene degradation. EbN1 also degrades various other aromatic and phenolic compounds in the absence of oxygen, one of them being p-ethylphenol. Despite having similar chemical structures, ethylbenzene and p-ethylphenol have been proposed to be metabolized by completely separate pathways. In this study, we established and applied biochemical and molecular biological methods to show the (almost) exclusive presence and specificity of enzymes involved in the respective degradation pathways by recording enzyme activities, complemented by heme staining, immuno- and biotin-blotting analyses. These combined results substantiated the predicted p-ethylphenol degradation pathway. The identified enzymes include a heme c-containing p-ethylphenol-hydroxylase, both an (R)- and an (S)-specific alcohol dehydrogenase as well as a novel biotin-dependent carboxylase. We also establish an activity assay for benzoylacetate-CoA ligases likely being involved in both metabolic pathways.

Coordination of Synthesis and Assembly of a Modular Membrane-Associated [NiFe]-Hydrogenase Is Determined by Cleavage of the C-Terminal Peptide.

UNLABELLED: During biosynthesis of [NiFe]-hydrogenase 2 (Hyd-2) of Escherichia coli, a 15-amino-acid C-terminal peptide is cleaved from the catalytic large subunit precursor, pro-HybC. This peptide is removed only after NiFe(CN)2CO cofactor insertion by the Hyp accessory protein machinery has been completed, suggesting that it has a regulatory function during enzyme maturation. We show here that in hyp mutants that fail to synthesize and insert the NiFe cofactor, and therefore retain the peptide, the Tat (twin-arginine translocon) signal peptide on the small subunit HybO is not removed and the subunit is degraded. In a mutant lacking the large subunit, the Tat signal peptide was also not removed from pre-HybO, indicating that the mature large subunit must actively engage the small subunit to elicit Tat transport. We validated the proposed regulatory role of the C-terminal peptide in controlling enzyme assembly by genetically removing it from the precursor of HybC, which allowed assembly and Tat-dependent membrane association of a HybC-HybO heterodimer lacking the NiFe(CN)2CO cofactor. Finally, genetic transfer of the C-terminal peptide from pro-HyaB, the large subunit of Hyd-1, onto HybC did not influence its dependence on the accessory protein HybG, a HypC paralog, or the specific protease HybD. This indicates that the C-terminal peptide per se is not required for interaction with the Hyp machinery but rather suggests a role of the peptide in maintaining a conformation of the protein suitable for cofactor insertion. Together, our results demonstrate that the C-terminal peptide on the catalytic subunit controls biosynthesis, assembly, and membrane association of Hyd-2. IMPORTANCE: [NiFe]-hydrogenases are multisubunit enzymes with a catalytic subunit containing a NiFe(CN)2CO cofactor. Results of previous studies suggested that after synthesis and insertion of the cofactor by the Hyp accessory proteins, this large subunit changes conformation upon proteolytic removal of a short peptide from its C terminus. We show that removal of this peptide is necessary to allow the cleavage of the Tat signal peptide from the small subunit with concomitant membrane association of the heterodimer to occur. Genetic removal of the C-terminal peptide from the large subunit allowed productive interaction with the small subunit and Tat-dependent membrane insertion of a NiFe cofactor-free enzyme. Results based on swapping of C-terminal peptides between hydrogenases suggest that this peptide governs enzyme assembly via a conformational switch.

A Fluorescent Bioreporter for Acetophenone and 1-Phenylethanol derived from a Specifically Induced Catabolic Operon.

The ß-proteobacterium Aromatoleum aromaticum degrades the aromatic ketone acetophenone, a key intermediate of anaerobic ethylbenzene metabolism, either aerobically or anaerobically via a complex ATP-dependent acetophenone carboxylase and a benzoylacetate-CoA ligase. The genes coding for these enzymes (apcABCDE and bal) are organized in an apparent operon and are expressed in the presence of the substrate acetophenone. To study the conditions under which this operon is expressed in more detail, we constructed a reporter strain by inserting a gene fusion of apcA, the first gene of the apc-bal operon, with the gene for the fluorescent protein mCherry into the chromosome of A. aromaticum. The fusion protein indeed accumulated consistently with the expression pattern of the acetophenone-metabolic enzymes under various growth conditions. After evaluating and quantifying the data by fluorescence microscopy, fluorescence-based flow cytometry and immunoblot analysis, mCherry production was found to be proportional to the applied acetophenone concentrations. The reporter strain allowed quantification of acetophenone within a concentration range of 50 µM (detection limit) to 250 µM after 12 and 24 h. Moreover, production of the Apc-mCherry fusion protein in the reporter strain was highly specific and responded to acetophenone and both enantiomers of 1-phenylethanol, which are easily converted to acetophenone. Other analogous substrates showed either a significantly weaker response or none at all. Therefore, the reporter strain provides a basis for the development of a specific bioreporter system for acetophenone with an application potential reaching from environmental monitoring to petroleum prospecting.

[NiFe]-hydrogenase maturation: isolation of a HypC-HypD complex carrying diatomic CO and CN- ligands.

The HypC and HypD maturases are required for the biosynthesis of the Fe(CN)(2)CO cofactor in the large subunit of [NiFe]-hydrogenases. Using infrared spectroscopy we demonstrate that an anaerobically purified, Strep-tagged HypCD complex from Escherichia coli exhibits absorption bands characteristic of diatomic CO and CN(-) ligands as well as CO(2). Metal and sulphide analyses revealed that along with the [4Fe-4S](2+) cluster in HypD, the complex has two additional oxygen-labile Fe ions. We prove that HypD cysteine 41 is required for the coordination of all three ligands. These findings suggest that the HypCD complex carries minimally the Fe(CN)(2)CO cofactor.

Evidence for an oxygen-sensitive iron-sulfur cluster in an immature large subunit species of Escherichia coli [NiFe]-hydrogenase 2.

Endoprotease-specific C-terminal processing is required to complete the maturation of the large subunit of [NiFe]-hydrogenases. This happens only after synthesis and insertion of the NiFe(CN)(2)CO cofactor by the Hyp maturases has occurred. It is assumed that in the absence of maturation the unprocessed species of the large subunit lacks cofactors. In this study we isolated a variant of the hydrogenase 2 large subunit, HybC, containing a fused C-terminal pentapeptide. The polypeptide could not be processed and was unable to associate with the small subunit to deliver an active enzyme. The His(6)-HybC variant protein isolated was brown and had sub-stoichiometric amounts of an oxygen-sensitive Iron-sulfur cluster, which could be chemically reconstituted to a [4Fe-4S] cluster. This cluster was coordinated by the conserved cysteinyl residues that normally ligate the NiFe(CN)(2)CO cofactor. Our findings provide evidence for temporary promiscuity of cofactor-binding sites.