Activation of short-chain ketones and isopropanol in sulfate-reducing bacteria.

BACKGROUND: Degradation of acetone by aerobic and nitrate-reducing bacteria can proceed via carboxylation to acetoacetate and subsequent thiolytic cleavage to two acetyl residues. A different strategy was identified in the sulfate-reducing bacterium Desulfococcus biacutus that involves formylation of acetone to 2-hydroxyisobutyryl-CoA. RESULTS: Utilization of short-chain ketones (acetone, butanone, 2-pentanone and 3-pentanone) and isopropanol by the sulfate reducer Desulfosarcina cetonica was investigated by differential proteome analyses and enzyme assays. Two-dimensional protein gel electrophoresis indicated that D. cetonica during growth with acetone expresses enzymes homologous to those described for Desulfococcus biacutus: a thiamine diphosphate (TDP)-requiring enzyme, two subunits of a B12-dependent mutase, and a NAD+-dependent dehydrogenase. Total proteomics of cell-free extracts confirmed these results and identified several additional ketone-inducible proteins. Acetone is activated, most likely mediated by the TDP-dependent enzyme, to a branched-chain CoA-ester, 2-hydroxyisobutyryl-CoA. This compound is linearized to 3-hydroxybutyryl-CoA by a coenzyme B12-dependent mutase followed by oxidation to acetoacetyl-CoA by a dehydrogenase. Proteomic analysis of isopropanol- and butanone-grown cells revealed the expression of a set of enzymes identical to that expressed during growth with acetone. Enzyme assays with cell-free extract of isopropanol- and butanone-grown cells support a B12-dependent isomerization. After growth with 2-pentanone or 3-pentanone, similar protein patterns were observed in cell-free extracts as those found after growth with acetone. CONCLUSIONS: According to these results, butanone and isopropanol, as well as the two pentanone isomers, are degraded by the same enzymes that are used also in acetone degradation. Our results indicate that the degradation of several short-chain ketones appears to be initiated by TDP-dependent formylation in sulfate-reducing bacteria.

Enzymes involved in phthalate degradation in sulphate-reducing bacteria.

The complete degradation of the xenobiotic and environmentally harmful phthalate esters is initiated by hydrolysis to alcohols and o-phthalate (phthalate) by esterases. While further catabolism of phthalate has been studied in aerobic and denitrifying microorganisms, the degradation in obligately anaerobic bacteria has remained obscure. Here, we demonstrate a previously overseen growth of the δ-proteobacterium Desulfosarcina cetonica with phthalate/sulphate as only carbon and energy sources. Differential proteome and CoA ester pool analyses together with in vitro enzyme assays identified the genes, enzymes and metabolites involved in phthalate uptake and degradation in D. cetonica. Phthalate is initially activated to the short-lived phthaloyl-CoA by an ATP-dependent phthalate CoA ligase (PCL) followed by decarboxylation to the central intermediate benzoyl-CoA by an UbiD-like phthaloyl-CoA decarboxylase (PCD) containing a prenylated flavin cofactor. Genome/metagenome analyses predicted phthalate degradation capacity also in the sulphate-reducing Desulfobacula toluolica, strain NaphS2, and other δ-proteobacteria. Our results suggest that phthalate degradation proceeds in all anaerobic bacteria via the labile phthaloyl-CoA that is captured and decarboxylated by highly abundant PCDs. In contrast, two alternative strategies have been established for the formation of phthaloyl-CoA, the possibly most unstable CoA ester in biology.

Four Molybdenum-Dependent Steroid C-25 Hydroxylases: Heterologous Overproduction, Role in Steroid Degradation, and Application for 25-Hydroxyvitamin D3 Synthesis.

Side chain-containing steroids are ubiquitous constituents of biological membranes that are persistent to biodegradation. Aerobic, steroid-degrading bacteria employ oxygenases for isoprenoid side chain and tetracyclic steran ring cleavage. In contrast, a Mo-containing steroid C-25 dehydrogenase (S25DH) of the dimethyl sulfoxide (DMSO) reductase family catalyzes the oxygen-independent hydroxylation of tertiary C-25 in the anaerobic, cholesterol-degrading bacterium Sterolibacterium denitrificans Its genome contains eight paralogous genes encoding active site α-subunits of putative S25DH-like proteins. The difficult enrichment of labile, oxygen-sensitive S25DH from the wild-type bacteria and the inability of its active heterologous production have largely hampered the study of S25DH-like gene products. Here we established a heterologous expression platform for the three structural genes of S25DH subunits together with an essential chaperone in the denitrifying betaproteobacterium Thauera aromatica K172. Using this system, S25DH1 and three isoenzymes (S25DH2, S25DH3, and S25DH4) were overproduced in a soluble, active form allowing a straightforward purification of nontagged αßγ complexes. All S25DHs contained molybdenum, four [4Fe-4S] clusters, one [3Fe-4S] cluster, and heme B and catalyzed the specific, water-dependent C-25 hydroxylations of various 4-en-3-one forms of phytosterols and zoosterols. Crude extracts from T. aromatica expressing genes encoding S25DH1 catalyzed the hydroxylation of vitamin D3 (VD3) to the clinically relevant 25-OH-VD3 with >95% yield at a rate 6.5-fold higher than that of wild-type bacterial extracts; the specific activity of recombinant S25DH1 was twofold higher than that of wild-type enzyme. These results demonstrate the potential application of the established expression platform for 25-OH-VD3 synthesis and pave the way for the characterization of previously genetically inaccessible S25DH-like Mo enzymes of the DMSO reductase family.IMPORTANCE Steroids are ubiquitous bioactive compounds, some of which are considered an emerging class of micropollutants. Their degradation by microorganisms is the major process of steroid elimination from the environment. While oxygenase-dependent steroid degradation in aerobes has been studied for more than 40 years, initial insights into the anoxic steroid degradation have only recently been obtained. Molybdenum-dependent steroid C25 dehydrogenases (S25DHs) have been proposed to catalyze oxygen-independent side chain hydroxylations of globally abundant zoo-, phyto-, and mycosterols; however, so far, their lability has allowed only the initial characterization of a single S25DH. Here we report on a heterologous gene expression platform that allowed for easy isolation and characterization of four highly active S25DH isoenzymes. The results obtained demonstrate the key role of S25DHs during anoxic degradation of various steroids. Moreover, the platform is valuable for the efficient enzymatic hydroxylation of vitamin D3 to its clinically relevant C-25-OH form.

Evolution of a xenobiotic degradation pathway: formation and capture of the labile phthaloyl-CoA intermediate during anaerobic phthalate degradation.

Xenobiotic phthalates are industrially produced on the annual million ton scale. The oxygen-independent enzymatic reactions involved in anaerobic phthalate degradation have only recently been elucidated. In vitro assays suggested that phthalate is first activated to phthaloyl-CoA followed by decarboxylation to benzoyl-CoA. Here, we report the heterologous production and characterization of the enzyme initiating anaerobic phthalate degradation from 'Aromatoleum aromaticum': a highly specific succinyl-CoA:phthalate CoA transferase (SPT, class III CoA transferase). Phthaloyl-CoA formed by SPT accumulated only to sub-micromolar concentrations due to the extreme lability of the product towards intramolecular substitution with a half-life of around 7 min. Upon addition of excess phthaloyl-CoA decarboxylase (PCD), the combined activity of both enzymes was drastically shifted towards physiologically relevant benzoyl-CoA formation. In conclusion, a massive overproduction of PCD in phthalate-grown cells to concentrations >140 µM was observed that allowed for efficient phthaloyl-CoA conversion at concentrations 250-fold below the apparent Km -value of PCD. The results obtained provide insights into an only recently evolved xenobiotic degradation pathway where a massive cellular overproduction of PCD compensates for the formation of the probably most unstable CoA ester intermediate in biology.

Reversibility of citrate synthase allows autotrophic growth of a thermophilic bacterium.

Biological inorganic carbon fixation proceeds through a number of fundamentally different autotrophic pathways that are defined by specific key enzymatic reactions. Detection of the enzymatic genes in (meta)genomes is widely used to estimate the contribution of individual organisms or communities to primary production. Here we show that the sulfur-reducing anaerobic deltaproteobacterium Desulfurella acetivorans is capable of both acetate oxidation and autotrophic carbon fixation, with the tricarboxylic acid cycle operating either in the oxidative or reductive direction, respectively. Under autotrophic conditions, the enzyme citrate synthase cleaves citrate adenosine triphosphate independently into acetyl coenzyme A and oxaloacetate, a reaction that has been regarded as impossible under physiological conditions. Because this overlooked, energetically efficient carbon fixation pathway lacks key enzymes, it may function unnoticed in many organisms, making bioinformatical predictions difficult, if not impossible.

A patchwork pathway for oxygenase-independent degradation of side chain containing steroids.

The denitrifying betaproteobacterium Sterolibacterium denitrificans serves as model organism for studying the oxygen-independent degradation of cholesterol. Here, we demonstrate its capability of degrading various globally abundant side chain containing zoo-, phyto- and mycosterols. We provide the complete genome that empowered an integrated genomics/proteomics/metabolomics approach, accompanied by the characterization of a characteristic enzyme of steroid side chain degradation. The results indicate that individual molybdopterin-containing steroid dehydrogenases are involved in C25-hydroxylations of steroids with different isoprenoid side chains, followed by the unusual conversion to C26-oic acids. Side chain degradation to androsta-1,4-diene-3,17-dione (ADD) via aldolytic C-C bond cleavages involves acyl-CoA synthetases/dehydrogenases specific for the respective 26-, 24- and 22-oic acids/-oyl-CoAs and promiscuous MaoC-like enoyl-CoA hydratases, aldolases and aldehyde dehydrogenases. Degradation of rings A and B depends on gene products uniquely found in anaerobic steroid degraders, which after hydrolytic cleavage of ring A, again involves CoA-ester intermediates. The degradation of the remaining CD rings via hydrolytic cleavage appears to be highly similar in aerobic and anaerobic bacteria. Anaerobic cholesterol degradation employs a composite repertoire of more than 40 genes partially known from aerobic degradation in gammaproteobacteria/actinobacteria, supplemented by unique genes that are required to circumvent oxygenase-dependent reactions.

Phthaloyl-coenzyme A decarboxylase from Thauera chlorobenzoica: the prenylated flavin-, K+ - and Fe2+ -dependent key enzyme of anaerobic phthalate degradation.

The degradation of the industrially produced and environmentally relevant phthalate esters by microorganisms is initiated by the hydrolysis to alcohols and phthalate (1,2-dicarboxybenzene). In the absence of oxygen the further degradation of phthalate proceeds via activation to phthaloyl-CoA followed by decarboxylation to benzoyl-CoA. Here, we report on the first purification and characterization of a phthaloyl-CoA decarboxylase (PCD) from the denitrifying Thauera chlorobenzoica. Hexameric PCD belongs to the UbiD-family of (de)carboxylases and contains prenylated FMN (prFMN), K+ and, unlike other UbiD-like enzymes, Fe2+ as cofactors. The latter is suggested to be involved in oxygen-independent electron-transfer during oxidative prFMN maturation. Either oxidation to the Fe3+ -state in air or removal of K+ by desalting resulted in >92% loss of both, prFMN and decarboxylation activity suggesting the presence of an active site prFMN/Fe2+ /K+ -complex in PCD. The PCD-catalysed reaction was essentially irreversible: neither carboxylation of benzoyl-CoA in the presence of 2 M bicarbonate, nor an isotope exchange of phthaloyl-CoA with 13 C-bicarbonate was observed. PCD differs in many aspects from prFMN-containing UbiD-like decarboxylases and serves as a biochemically accessible model for the large number of UbiD-like (de)carboxylases that play key roles in the anaerobic degradation of environmentally relevant aromatic pollutants.

An unusual strategy for the anoxic biodegradation of phthalate.

In the past two decades, the study of oxygen-independent degradation of widely abundant aromatic compounds in anaerobic bacteria has revealed numerous unprecedented enzymatic principles. Surprisingly, the organisms, metabolites and enzymes involved in the degradation of o-phthalate (1,2-dicarboxybenzene), mainly derived from phthalate esters that are annually produced at the million ton scale, are sparsely known. Here, we demonstrate a previously unknown capacity of complete phthalate degradation in established aromatic compound-degrading, denitrifying model organisms of the genera Thauera, Azoarcus and 'Aromatoleum'. Differential proteome analyses revealed phthalate-induced gene clusters involved in uptake and conversion of phthalate to the central intermediate benzoyl-CoA. Enzyme assays provided in vitro evidence for the formation of phthaloyl-CoA by a succinyl-CoA- and phthalate-specific CoA transferase, which is essential for the subsequent oxygen-sensitive decarboxylation to benzoyl-CoA. The extreme instability of the phthaloyl-CoA intermediate requires highly balanced CoA transferase and decarboxylase activities to avoid its cellular accumulation. Phylogenetic analysis revealed phthaloyl-CoA decarboxylase as a novel member of the UbiD-like, (de)carboxylase enzyme family. Homologs of the encoding gene form a phylogenetic cluster and are found in soil, freshwater and marine bacteria; an ongoing global distribution of a possibly only recently evolved degradation pathway is suggested.

Promiscuous Defluorinating Enoyl-CoA Hydratases/Hydrolases Allow for Complete Anaerobic Degradation of 2-Fluorobenzoate.

Biodegradation of the environmentally hazardous fluoroaromatics has mainly been associated with oxygenase-dependent defluorination reactions. Only very recently a novel mode of oxygen-independent defluorination was identified for the complete degradation of para-substituted fluoroaromatics in the denitrifying Thauera aromatica: a promiscuous class I benzoyl-coenzyme A (BzCoA) reductase (BCR) catalyzed the ATP-dependent defluorination of 4-F-BzCoA to BzCoA. Here, we studied the unknown enzymatic defluorination during the complete degradation of 2-F-benzoate to CO2 and HF. We demonstrate that after activation of 2-F-benzoate by a promiscuous AMP-forming benzoate-CoA ligase, the 2-F-BzCoA formed is subsequently dearomatized by BCR to a mixture of 2-F- and 6-F-cyclohexa-1,5-diene-1-carboxyl-CoA (2-F-/6-F-1,5-dienoyl-CoA). This finding indicates that BCR is not involved in C-F-bond cleavage during growth with 2-F-benzoate. Instead, we identified defluorination of the two isomers by enoyl-CoA hydratases/hydrolases involved in down-stream reactions of the BzCoA degradation pathway. (i) The 1,5-dienoyl-CoA hydratase hydrated the F-1,5-dienoyl-CoA isomers to a mixture of the stable 2-F-6-OH-1-enoyl-CoA and the unstable α-fluorohydrin 6-F-6-OH-1-enoyl-CoA; the latter spontaneously decomposed to HF and 6-oxo-cyclohex-1-enoyl-CoA (6-oxo-1-enoyl-CoA), a common intermediate of the BzCoA degradation pathway. (ii) 6-Oxo-1-enoyl-CoA hydrolase/hydratase catalyzed the defluorination of 2-F-6-OH-1-enoyl-CoA to 2-oxo-6-OH-1-enoyl-CoA and HF again via water addition to an F-enoyl-CoA functionality. Based on these in vitro results, we demonstrate a previously overseen capability of 2-F-benzoate degradation for many but not all tested facultatively and obligately anaerobic bacteria that degrade aromatic compounds via the BzCoA degradation pathway. In conclusion, the newly identified enzymatic defluorination by enoyl-CoA hydratases via α-fluorohydrin formation represents an abundant, physiologically relevant principle of enzymatic defluorination.

ATP-Dependent C-F Bond Cleavage Allows the Complete Degradation of 4-Fluoroaromatics without Oxygen.

UNLABELLED: Complete biodegradation of the abundant and persistent fluoroaromatics requires enzymatic cleavage of an arylic C-F bond, probably the most stable single bond of a biodegradable organic molecule. While in aerobic microorganisms defluorination of fluoroaromatics is initiated by oxygenases, arylic C-F bond cleavage has never been observed in the absence of oxygen. Here, an oxygen-independent enzymatic aryl fluoride bond cleavage is described during the complete degradation of 4-fluorobenzoate or 4-fluorotoluene to CO2 and HF in the denitrifying Thauera aromatica: the ATP-dependent defluorination of 4-fluorobenzoyl-coenzyme A (4-F-BzCoA) to benzoyl-coenzyme A (BzCoA) and HF, catalyzed by class I BzCoA reductase (BCR). Adaptation to growth with the fluoroaromatics was accomplished by the downregulation of a promiscuous benzoate-CoA ligase and the concomitant upregulation of 4-F-BzCoA-defluorinating/dearomatizing BCR on the transcriptional level. We propose an unprecedented mechanism for reductive arylic C-F bond cleavage via a Birch reduction-like mechanism resulting in a formal nucleophilic aromatic substitution. In the proposed anionic 4-fluorodienoyl-CoA transition state, fluoride elimination to BzCoA is favored over protonation to a fluorinated cyclic dienoyl-CoA. IMPORTANCE: Organofluorides are produced as pesticides, pharmaceuticals, and other chemicals and comprise approximately one quarter of all organic compounds in the pharmaceutical and agricultural sectors; they are considered a growing class of environmentally relevant persistent pollutants. Especially in the case of fluoroaromatics, biodegradation is hampered by the extreme stability of the arylic C-F bond. In aerobic microorganisms, degradation proceeds via oxygenase-dependent C-F bond cleavage reactions, whereas the enzymes involved in the degradation of fluoroaromatics at anoxic sites are unknown. Here we report a strategy for the complete biodegradation of a fluoroaromatic to CO2 and HF in a denitrifying bacterium via activation to a CoA ester, followed by oxygen-independent arylic C-F bond cleavage catalyzed by an ATP-dependent enzyme. This reaction, in conjunction with a transcriptional adaptation to fluorinated growth substrates, is essential for the anoxic biodegradation of 4-fluorobenzoate/4-F-toluene and probably other fluoroaromatics.

Enzymes involved in a novel anaerobic cyclohexane carboxylic acid degradation pathway.

The anaerobic degradation of cyclohexane carboxylic acid (CHC) has so far been studied only in Rhodopseudomonas palustris, in which CHC is activated to cyclohexanoyl coenzyme A (cyclohexanoyl-CoA [CHCoA]) and then dehydrogenated to cyclohex-1-ene-1-carboxyl-CoA (CHeneCoA). This intermediate is further degraded by reactions of the R. palustris-specific benzoyl-CoA degradation pathway of aromatic compounds. However, CHeneCoA is not an intermediate in the degradation of aromatic compounds in all other known anaerobic bacteria; consequently, degradation of CHC was mostly unknown in anaerobic bacteria. We identified a previously unknown CHC degradation pathway in the Fe(III)-reducing Geobacter metallireducens by determining the following CHC-induced in vitro activities: (i) the activation of CHC to CHCoA by a succinyl-CoA:CHC CoA transferase, (ii) the 1,2-dehydrogenation of CHCoA to CHeneCoA by CHCoA dehydrogenase, and (iii) the unusual 1,4-dehydrogenation of CHeneCoA to cyclohex-1,5-diene-1-carboxyl-CoA. This last represents a previously unknown joint intermediate of the CHC and aromatic compound degradation pathway in bacteria other than R. palustris. The enzymes catalyzing the three reactions were purified and characterized as specific enzymes after heterologous expression of the encoding genes. Quantitative reverse transcription-PCR revealed that expression of these genes was highly induced during growth with CHC but not with benzoate. The newly identified CHC degradation pathway is suggested to be present in nearly all CHC-degrading anaerobic bacteria, including denitrifying, Fe(III)-reducing, sulfate-reducing, and fermenting bacteria. Remarkably, all three CHC degradation pathways always link CHC catabolism to the catabolic pathways of aromatic compounds. We propose that the capacity to use CHC as a carbon source evolved from already-existing aromatic compound degradation pathways.