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Coenzyme B12‐Dependent 2‐Hydroxyisobutyryl‐Coa

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Coenzyme B12‐Dependent 2‐Hydroxyisobutyryl‐Coa Erschienen in: Environmental Microbiology Reports ; 10 (2018), 3. - S. 283-292 https://dx.doi.org/10.1111/1758-2229.12637 Two enzymes of the acetone degradation pathway of Desulfococcus biacutus: coenzyme B12-dependent 2-hydroxyisobutyryl-CoA mutase and 3-hydroxybutyryl-CoA dehydrogenase Jasmin Frey,1,2 Fabian Schneider,3 Thomas Huhn,3,4 and define an involvement of CoA esters in the path- Dieter Spiteller,1,4 Bernhard Schink1,4 and way. Further, the involvement of 2-hydroxyisobutyryl- 1,4 David Schleheck * CoA strongly indicates that the carbonyl-C2 of acetone 1Department of Biology, University of Konstanz, is added, most likely, to formyl-CoA through a TDP- D-78464 Konstanz, Germany. dependent enzyme that is co-induced in acetone- 2Graduate School Biological Sciences, University of grown cells and is encoded in the same gene cluster as Konstanz, D-78464 Konstanz, Germany. the identified mutase and dehydrogenase. 3Department of Chemistry, University of Konstanz, D-78464 Konstanz, Germany. 4Konstanz Research School Chemical Biology, Introduction University of Konstanz, D-78464 Konstanz, Germany. Acetone (dimethylketone) can be regarded as a partially acti- vated hydrocarbon through its oxo-group and is commonly Summary used in numerous industrial processes, e.g., as solvent or as Degradation of acetone by the sulfate-reducing bac- precursor for chemical syntheses. Furthermore, acetone is terium Desulfococcus biacutus involves an acetone- also produced by anaerobic, solventogenic bacteria like sev- activation reaction different from that used by aerobic eral Clostridia species (Durre€ et al., 1992; Sifniades et al., or nitrate-reducing bacteria, because the small 2011). Therefore, it occurs as an important pollutant in waste energy budget of sulfate-reducing bacteria does not water and also in natural habitats. Several degradation routes allow for major expenditures into ATP-consuming in an oxic environment are known, however, its biochemical carboxylation reactions. In the present study, an activation and utilization under anoxic conditions by sulfate- inducible coenzyme B12-dependent conversion of reducing bacteria (SRB) remained unclear until today. Two 2-hydroxyisobutyryl-CoA to 3-hydroxybutyryl-CoA was SRB of the Desulfosarcina/Desulfococcus cladehavebeen demonstrated in cell-free extracts of acetone-grown isolated and described that are able to utilize acetone as D. biacutus cells, together with a NAD1-dependent growth substrate, Desulfococcus biacutus strain KMRActS oxidation of 3-hydroxybutyryl-CoA to acetoacetyl- (DSM 5651) and Desulfosarcina cetonica strain 480 (DSM CoA. Genes encoding two mutase subunits and a 7267) (Platen et al., 1990; Janssen and Schink, 1995b). dehydrogenase, which were found previously to be While nitrate reducers usually activate acetone by a carboxyl- strongly induced during growth with acetone, were ation reaction forming acetoacetate, no acetone carboxylase heterologously expressed in E. coli. The activities of activity was detected in cell-free extracts of D. biacutus,and the purified recombinant proteins matched with the no acetone carboxylases were found in its genome and pro- inducible activities observed in cell-free extracts of teome (Janssen and Schink, 1995a; Gutierrez Acosta et al., acetone-grown D. biacutus: proteins (IMG locus tags) 2014). Initial studies suggested an involvement of carbon DebiaDRAFT_04573 and 04574 constituted a monoxide, and of acetoacetaldehyde rather than acetoace- tate, in the pathway (Gutierrez Acosta et al., 2013). Biochem- B12-dependent 2-hydroxyisobutyryl-CoA/3-hydroxy- butyryl-CoA mutase, and DebiaDRAFT_04571 was a ical studies with cell-free extracts indicated the involvement of 3-hydroxybutyryl-CoA dehydrogenase. Hence, these ATP and thiamine diphosphate (TDP) in acetone degradation (Gutierrez Acosta et al., 2014). However, genome sequenc- enzymes play key roles in the degradation of acetone ing and differential proteomics comparing acetone- and butyrate-grown cells (Gutierrez Acosta et al., 2014) identified several proteins that are specifically induced during growth with acetone. One of these (IMG locus tag Konstanzer Online-Publikations-System (KOPS) URL: http://nbn-resolving.de/urn:nbn:de:bsz:352-2-13g1kcwqkpciv5 284 Fig. 1. Hypothetical and confirmed reactions in D. biacutus for an acetone degradation pathway (A) that employs a yet inaccessible carbonyla- tion or formylation of acetone, and a 2-hydroxyisobutyryl-CoA mutase and 3-hydroxybutyryl-CoA dehydrogenase as identified in this study, and (B) illustration of the corresponding gene cluster in D. biacutus. A. The enzyme reactions identified in this study are the isomerization of 2-hydroxyisobutyryl-CoA to 3-hydroxybutyryl-CoA, which is catalyzed by a cofactor-B12 dependent methylmalonyl-CoA type mutase (red), and the subsequent oxidation of 3-hydroxybutyryl-CoA to acetoacetyl-CoA, which is catalyzed by a NAD1-dependent dehydrogenase (green). Subsequent reactions are cleavage of acetoacetyl-CoA into two acetyl-CoA, and the oxidation of acetyl-CoA via the reversed Wood-Ljungdahl pathway; the latter pathway is coupled to dissimilatory sulfate reduction (not shown) in D. biacutus. B. For the identified enzymes, the corresponding gene cluster and protein/gene identifiers are indicated (color coding) (IMG locus tags prefix, DebiaDRAFT_0), as well as for the co-encoded TDP-dependent enzyme, which is co-expressed during growth with acetone and which most likely is involved in an initial acetone carbonylation or formylation reaction (see question mark in A, and the text). DebiaDRAFT_04514) has recently been characterized as a considered likely, based on the preliminary data zinc-dependent alcohol dehydrogenase capable of converting described above. First, a direct carbonylation of the a wide range of aldehydes and alcohols (Frey et al., 2016). C2-carbon atom of acetone (shown as the lower route Further, in a different gene cluster, genes of four strongly in Fig. 1A) may produce a branched-chain aldehyde acetone-inducible proteins annotated as a predicted TDP- (2-hydroxy-2-methylpropanal), which may subsequently dependent enzyme (DebiaDRAFT_04566), two subunits of a be processed to a CoA ester. Second, addition of a for- B12-dependent methylmalonyl-CoA mutase-like enzyme myl residue, e.g., in form of formyl-CoA (shown in the (DebiaDRAFT_04573 and 04574), and a predicted short- upper route in Fig. 1A), could produce a branched-chain chain alcohol dehydrogenase (DebiaDRAFT_04573), were CoA ester intermediate, 2-hydroxyisobutyryl-CoA. The also identified by proteomics (Gutierrez Acosta et al., predicted B12-dependent methylmalonyl-CoA mutase-like 2014). The possible involvement of a B12-dependent enzyme would then isomerize the branched-chain CoA mutase led to the speculation whether a branched-chain ester to 3-hydroxybutyryl-CoA, which could be oxidized CoA ester may be formed during acetone activation, prior to acetoacetyl-CoA (Fig. 1A). to its conversion to, e.g., acetoacetyl-CoA (Gutierrez In the present study, a B12-dependent isomerization of Acosta et al., 2014). 2-hydroxyisobutyryl-CoA to 3-hydroxybutyryl-CoA was As illustrated in Fig. 1A, two routes of acetone activa- demonstrated, and an oxidation of 3-hydroxybutyryl-CoA tion leading to a branched-chain CoA ester were to acetoacetyl-CoA, both in cell-free extracts of D. 285 biacutus as well as with recombinant enzymes produced isomerizes 2-hydroxyisobutyryl-CoA to 3-hydroxybutyryl- from candidate genes attributed by differential proteo- CoA as part of an acetone-activating pathway in D. mics. The results strongly support the involvement of biacutus. these enzymes in acetone degradation, and further high- DebiaDRAFT_04571, which is strongly induced in light the potential role of a highly expressed TDP- acetone-grown cells (Gutierrez Acosta et al., 2014) and dependent enzyme for initial acetone activation. This encoded in the gene cluster as well (Fig. 1B), was anno- TDP-dependent enzyme is currently under investigation tated (IMG pipeline) as 3-oxoacyl-[acyl-carrier protein] in our lab, and will be addressed in a further study. reductase family of NAD(P)1-binding short-chain alcohol dehydrogenases (pfam00106) (Marchler-Bauer et al., Results 2014). Its amino acid sequence exhibited around 30% Sequence comparison and functional prediction sequence identity to characterized 3-hydroxybutyryl-CoA dehydrogenases/acetoacetyl-CoA reductases, e.g., of The amino acid sequence of DebiaDRAFT_04574 of D. Ralstonia solanacearum (PhaB, D8NBJ9), and the high- biacutus contains a conserved catalytic N-terminal est sequence identity (82%) was found for a dehydroge- domain of methylmalonyl-CoA mutase-like enzymes, nase candidate encoded in the gene cluster of D. which belong to a small family of coenzyme B12-depen- cetonica described above (Kim et al., 2014). Hence, dent isomerases (protein family pfam01642) (large subu- DebiaDRAFT_04571 represents a good candidate for a nit). The amino acid sequence of the neighboring locus conversion of 3-hydroxybutyryl-CoA to acetoacetyl-CoA, tag, DebiaDRAFT_04573, is predicted to represent the which is postulated to be formed during acetone corresponding coenzyme B -binding small subunit of 12 activation. methylmalonyl-CoA mutases (pfam02310) (Banerjee and Ragsdale, 2003). A sequence comparison across all cur- 2-Hydroxyisobutyryl-CoA mutase activity detected in rently available bacterial genomes (as per November cell-free extract of acetone-grown cells 2017; NCBI BLAST) revealed for both subunits highest We synthesized
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