Characterization of a novel enzyme system for activation of acetone in sulfate-reducing bacteria
Microbial Ecology and Applied Microbiology
Final Report Abstract
The capacity for acetone degradation in sulfate-reducing bacteria was found originally in Desulfococcus biacutus and later in Desulfosarcina cetonica. Both contained a similar gene cluster comprising genes encoding a TDP-dependent α-acetolactate synthase-like enzyme, a coenzyme B12-dependent 2-hydroxyisobutyryl-CoA: 3-hydroxybutyryl-CoA isomerase, and an NAD-dependent 3-hydroxybutyryl-CoA dehydrogenase. The B12-dependent isomerase and the dehydrogenase were cloned and heterologously expressed in E. coli. We identified the same gene clusters and the same metabolic capacity also in two further sulfate reducers, Desulfallas (formerly Desulfotomaculum) arcticus and Desulfallas geothermicus. These findings indicate that possession of this gene cluster is crucial for the metabolic capability of acetone degradation in sulfate-reducing microorganisms, and that acetone degradation in all these cases proceeds via 2-hydroxyisobutyryl CoA as a central intermediate. This rules out the hypothetical alternative pathway via 3-hydroxybutyraldehyde. Numerous efforts to show the activity of a TDP-dependent enzyme converting acetone with an activated one-carbon compound to 2-hydroxyisobutyryl-CoA in cell-free extracts failed. Since according to gene sequence data this enzyme resembles α-acetolactate synthase and other TDP-dependent one-carbon metabolizing enzymes such as pyruvate formate lyase we tried numerous modifications of our original enzyme assay, varying the buffer system, reducing agent, one-carbon donor, the measurement concept and HPLC detector systems, and also tried to reactivate the enzyme by preincubations with Fe(II) and sulfide or with enhanced background protein concentrations. All these efforts failed, and therefore we cannot completely identify the co-substrate(s), despite the fact that we can identify the enzyme at the level of protein primary structure given by the specifically induced acetone degradation genes. Efforts to purify the acetone-fermenting partner in methanogenic acetone-degrading enrichment cultures were not successful yet. Comparison of 16SrRNA sequences showed that it is probably related to an incompletely oxidizing sulfate reducer, Desulfomicrobium baculatum. Metaproteome and metagenome analysis of this methanogenic enrichment culture indicated that it contains the same key enzymes mentioned above that appear to be essential for acetone degradation by sulfate-reducing bacteria.
Publications
- Synthesis of short-chain hydroxyaldehydes and their 2,4-dinitrophenylhydrazone derivatives, and separation of their isomers by HPLC. J. Chromatogr. Ser. A 1531, 143–150 (2018)
Frey, J., F. Schneider, B. Schink, T. Huhn
(See online at https://doi.org/10.1016/j.chroma.2017.11.046) - Two enzymes of the acetone degradation pathway of Desulfococcus biacutus: coenzyme B12-dependent 2-hydroxyisobutyryl-CoA mutase and 3-hydroxybutyryl-CoA dehydrogenase. Environ. Microbiol. Rep. 10, 283-292 (2018)
Frey, J., F. Schneider, T. Huhn, D. Spiteller, B. Schink, D. Schleheck
(See online at https://doi.org/10.1111/1758-2229.12637)
