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Biotransformation of monoterpenes, bile acids, and other isoprenoids in anaerobic ecosystems

P.B. Hylemon, J. Harder
DOI: http://dx.doi.org/10.1111/j.1574-6976.1998.tb00382.x 475-488 First published online: 1 December 1998


Isoprenoic compounds play a major part in the global carbon cycle. Biosynthesis and mineralization by aerobic bacteria have been intensively studied. This review describes our knowledge on the anaerobic metabolism of isoprenoids, mainly by denitrifying and fermentative bacteria. Nitrate-reducing β-Proteobacteria were isolated on monoterpenes as sole carbon source and electron donor. Thauera spp. were obtained on the oxygen-containing monoterpenes linalool, menthol, and eucalyptol. Several strains of Alcaligenes defragrans were isolated on unsaturated monoterpenes as growth substrates. A novel denitrifying β-Proteobacterium, strain 72Chol, mineralizes cholesterol completely to carbon dioxide. Physiological studies showed the presence of several oxidative pathways in these microorganisms. Investigations by organic geochemists indicate possible contributions of anaerobes to early diagenetic processes. One example, the formation of p-cymene from monoterpenes, could indeed be detected in methanogenic enrichment cultures. In man, cholic acid (CA) and chenodeoxycholic acid (CDCA), are synthesized in the liver from cholesterol. During their enterohepatic circulation, bile acids are biotransformed by the intestinal microflora into a variety of metabolites. Known bacterial biotransformations of conjugated bile acids include: deconjugation, oxidation of hydroxy groups at C-3, C-7 and C-12 with formation of oxo bile acids and reduction of these oxo groups to either α- or β-configuration. Quantitatively, the most important bacterial biotransformation is the 7α-dehydroxylation of CA and CDCA yielding deoxycholic acid and lithocholic acid, respectively. The 7α-dehydroxylation of CA occurs via a novel six-step biochemical pathway. The genes encoding several enzymes that either transport bile acids or catalyze various reactions in the 7α-dehydroxylation pathway of Eubacterium sp. strain VPI 12708 have been cloned, expressed in Escherichia coli, purified, and characterized.

  • 7α-Dehydroxylation
  • Intestinal bacteria
  • Cholesterol

1 Isoprenoic compounds in nature

Living organisms synthesize a remarkable diversity of isoprenoids. More than 23 000 different compounds have been isolated and all contain one or more isoprene unit, the building block of these compounds. An isoprene unit is a 5-carbon unit, 2-methyl-butane, derived from isopentyl pyrophosphate and formed via two alternative pathways, the mevalonate (in eukaryotes) and the deoxyxylulose pathway in prokaryotes and plant plastids [1,2].

The formed isoprenoids present a large fraction of the organic matter on earth. Aerobic bacteria utilize these compounds as electron donor and carbon source [36]. However, isoprenoic chemical structures, e.g. sterols, might be considered recalcitrant towards degradation due to the low number of functional groups and the complex carbon skeleton. Aerobic bacteria circumvent this problem by oxygenation reactions with molecular oxygen as cosubstrate. Anaerobic bacteria must apply a different biochemical strategy. This review reports our knowledge on the anaerobic metabolism of isoprenoids and sterols by microbes in anaerobic ecosystems.

1.1 Monoterpenes

Monoterpenes are formed almost exclusively in plants. Terpene emission from trees is estimated at 4.8×1014 g per year [7]. Leaves may contain up to 9% w/w monoterpenes per dry weight [8] and contribute as fall foliage to the organic matter in soils and sediments. The absence of monoterpenes in aged sediment layers [9] was for a long time ascribed to diffusion into the atmosphere. The solubility of monoterpene hydrocarbons, 20–50 μM in water [10], is sufficient to remove these compounds by diffusion with time from the subsurface. Additionally, monoterpenes are toxic for cells and hence are believed to purvey a plant defense against herbivores [11].

1.2 Cell membrane compounds

Membranes hold several classes of isoprenoids, e.g. carotinoids, polyprenols and sterols. These lipophilic substances have extremely low solubilities in water. Once buried below the sediment surface, the molecules have a good chance to endure due to low diffusive rates which limit mainly the microbial degradation. Most sediment organisms are immobile and rely on diffusive or convective transport of the substrates for metabolic activity [12]. Hence, anaerobic habitats with a significant convection or short diffusive distances, i.e. the intestinal system and denitrifying activated sludge, may be appropriate for metabolic studies of large isoprenoid compounds.

2 Degradation of monoterpenes by denitrifying bacteria

The chemistry of monoterpenes is based on an huge diversity of structural isomers and involves intricate stereochemical problems and bizarre rearrangement reactions [13]. The study of monoterpene biosynthesis has clearly contributed to our understanding of unusual enzymes, in particular the monoterpene synthases, and the enzymatic control of reaction pathways [14,15]. The study of monoterpene degradation in the absence of molecular oxygen may allow the discovery of novel biotransformations of several unique structural units, e.g. tertiary alcohols, alkenes and bicyclic compounds. Recent research identified many monoterpenes that support denitrifying growth of enrichment cultures or pure strains (Table 1) ([1618], J. Harder, unpublished). The ecological importance of these traits was tested by most-probable-number (MPN) determinations of the population size. Statistically, each culturable denitrifying bacterium present in soil under a coniferous tree had the ability to degrade monoterpene alkenes (107 cells (ml soil)−1). In sewage sludge, one of 150 denitrifying microbes was able to grow on monoterpenes (106 cells (ml sludge)−1) [19]. The physiological characterization of isolated strains of β-Proteobacteria provided the means to identify degradative pathways for some monoterpenes.

View this table:
Table 1

Monoterpenes that are sole organic growth substances of denitrifying microorganisms

Monoterpenes utilized by isolated strains
Alkenes(+)-2-carene, (+)-3-carene, (+)-limonene, (+)-p-menth-1-ene, myrcene, (−)-α-phellandrene, (−)-α-pinene, (−)-β-pinene, (+)-sabinene, α-terpinene, γ-terpinene, terpinolene
Oxygen containing compounds(−)-β-citronellol, 3,7-dimethyloctanol-1, eucalyptol, geraniol, (+)-isomenthol, (+)-isopulegol, linalool, menthol, menthone, (+)-pulegone, (+)-α-terpineol, (+)-terpinen-4-ol
Monoterpenes utilized by enrichment cultures
Alkenes(−)-camphene, (−)-β-citronellene, (+)-trans-isolimonene, ocimene
Oxygen containing compounds(−)-borneol, (+)-camphor, (+)-camphoric acid, (−)-carvone, 1,4-cineol, (+)-fenchol, (+)-fenchone, (+)-trans-myrtanol, (−)-myrtenol, nerol, (+)-perilla alcohol, α+β-thujone, cis-verbenol, (−)-verbenone

2.1 Linalool and other aliphatic substances

Methyl groups of branched compounds, e.g. 3,7-dimethyl-octanol-1, impede the utilization of the β-oxidation pathway of fatty acids. Early studies by Seubert et al. described the carboxylation of the β-methyl-α,β-en-acid CoA thioester that enables a removal of the methyl group as acetate [4,20,21]. Because this pathway is independent of molecular oxygen, it may also be present in anaerobic microorganisms. Pseudomonas citronellolis could in fact grow anaerobically on either 3,7-dimethyl-octanol-1 or citronellol and nitrate [16]. The mineralization of a tertiary alcohol was investigated with linalool, an allyl alcohol. The isolated denitrifying bacterium, Thauera linaloolentis[16,17], isomerizes linalool regioselectively to geraniol, thus without formation of nerol (Fig. 1) [22]. Analysis of the macroscopic 1,3-hydroxyl-Δ21-mutase activity established that linalool is 5.9 kJ mol−1 more stable than geraniol. The transient formation of geranial provided evidence for a further degradation similar to citronellol [22].

Figure 1

Initial pathway of linalool degradation.

2.2 Menthol

Thauera terpenica strain 21Mol grows on (−)-menthol, (−)-menthone, (+)-isomenthol, (−)-isopulegol and (+)-pulegone, which carry all an oxygen-functional group at the C-3 atom (menthane nomenclature) [17]. Other menthane-derived alcohols, e.g. (+)-α-terpineol or (+)-terpinen-4-ol, are not utilized. Therefore, an oxygenated C-3 atom seems to be essential for the mineralization of menthol. This fosters the hypothesis that menthol is oxidized in a manner similar to cyclohexanol on a β-cleavage pathway (Fig. 2) [23,24]. All compounds utilized may initially be oxidized or rearranged to menth-2-enone which may undergo a water addition. After oxidation to mentha-1,3-dione, a thiolytic attack may open the carbon ring, and the product 3,7-dimethyl-5-oxo-octane acid CoA thioester may be degraded on Seubert’s pathway. Additional evidence for this hypothesis came from growth of strain 21Mol on 3,7-dimethyl-octanol-1 [17].

Figure 2

Proposed ring cleavage of menthol.

2.3 Eucalyptol

Ether cleavages can occur with several pathways [25]; an alcohol elimination with a concomitant formation of an alkene was only reported for the cleavage of O-carboxymethyl-malate into glycolate and fumarate [26]. This reaction is a model for the initial reaction of eucalyptol degradation by Thauera terpenica strain 58Eu that was isolated on eucalyptol and nitrate (Fig. 3) [16,17]. Growth substrates of this bacterium include the potential cleavage product, α-terpineol, and some other alkenoic monoterpenes [17].

Figure 3

Proposed initial reactions of eucalyptol mineralization.

2.4 Monoterpene alkenes

The isolation of denitrifying bacteria on alkenoic monoterpenes provided several Alcaligenes defragrans strains [16,18]. These microbes differ in their monoterpene utilization patterns from Thauera terpenica strain 58Eu: the former exhibit no growth on eucalyptol, whereas the later grew on myrcene, (+)-menth-1-ene, (+)-2-carene, (+)-3-carene and (−)-α-pinene. Both species utilized the alcohols α-terpineol and (+)-terpinen-4-ol, and the alkenes terpinolene, α-terpinene, γ-terpinene, (−)-α-phellandrene, (+)-limonene, (+)-sabinene and (−)-β-pinene [17,18]. Interestingly, the C-1 atom (according to the menthane nomenclature) is in all compounds sp2-hybridized. Whether this hybridization is of importance was studied with (+)-trans-isolimonene which has an sp3-hybridized C-1 atom. A quantitative biotransformation of isolimonene into isoterpinolene was performed by A. defragrans strains in the presence of a monoterpene as a growth substrate, but not by Thauera terpenica strain 58Eu (Fig. 4) [75]. Menthadienes were formed in small traces in cultures growing on bicyclic compounds [16]. Hence these bacteria have enzymes that rearrange unsaturated monoterpene – a reaction occurring abiotically at elevated temperatures. The C-1 sp2 hybridization of menthadienes seems to be essential for the further metabolism in which the monoterpene may be transformed into an ionic compound that stays intracellular as substrate.

Figure 4

Isomerization of isolimonene to isoterpinolene.

3 Metabolism of monoterpenes in methanogenic habitats

One of the rare encounters with monoterpenes in geological samples were peat-covered pine stumps [27]. The amount of α-pinene decreased with increasing depth of burial and went with an increase of the content of p-cymene and p-menthane. This disproportionation into alkanes and aromatics with an intact carbon skeleton is typical for diagenetic processes and raises the question of a biogenic contribution. The formation of p-cymene from 2-carene, 3-carene and α-pinene in denitrifying enrichment cultures was a first hint for a microbial origin of p-cymene [16]. Miyazawa et al. [28] reported recently the formation of p-cymene and mentha-1,3-dien-7-ol from α-terpinene by anaerobically incubated intestinal bacteria of the common cutworm larvae. However, the formation of a typical monooxygenation product, mentha-1,3-dien-7-ol, questions the anaerobiosis in these experiments.

When sewage sludge was tested under methanogenic conditions for the ability to aromatize some monoterpenes, sabinene and α-phellandrene, but not α-pinene and 2-carene were quantitatively transformed to p-cymene [19]. In the first subculture, sabinene was metabolized to equal amounts of γ-terpinene and p-cymene. This indicates a rearrangement of sabinene to a menthadiene (Fig. 5). In contrast to sabinene, the transformation of α-phellandrene deceased in the first subculture. This may be due to the free energy change of the aromatization reaction to p-cymene and molecular hydrogen: it is with a value of −18 kJ mol−1 less for α-phellandrene than for sabinene (ΔG°=−129.8 kJ mol−1). The bacterial p-cymene synthesis from monoterpenes presents a different route to natural aromatics in comparison to the formation of toluene from aromatic amino acids by fermenting bacteria, e.g. Tolumonas auensis[29,30].

Figure 5

Proposed pathway of p-cymene formation.

4 Organic geochemistry and anaerobic microbiology of cell membrane compounds

Both aerobic and anaerobic microorganisms contribute to the early diagenesis of organic matter in sediments. Organic geochemical analyses are retrospective views on the history of organic matter and may supply hints on transformations of sesqui-, tri- and tetraterpenoids by anaerobic microbes.

The side-chain of chlorophyll, phytol, is one of the most common isoprenoids in nature and most likely the precursor of phytane and pristane in sediments [27]. The recent report of pristane degradation in denitrifying microcosms and enrichment cultures [31] indicates that anaerobic alkane degradation is not limited to n-alkanes [3234].

Another phytol-derived early diagenic compound, 6,10,14-trimethylpentadecan-2-one, is metabolized by a denitrifying bacterium [35] and in sulfate-reducing enrichment cultures [36]. The degradative problem of this compound, the activation of an β-methyl ketone, may occur by carboxylation similar to acetone [37] and acetophenone [38]. Squalene served as sole electron donor and carbon source in methanogenic [39] and denitrifying enrichments (J. Harder, unpublished). A β-Proteobacterium, strain 72Chol, exhibited denitrifying activity on squalene [40]. Pigments of phototrophic organisms are degraded in anoxic sediments [41]. This phenomenon was also observed in laboratory incubations, e.g. a loss of β-carotene and xanthins occurred in anaerobic incubations of anoxic lake water samples [42]. Microorganisms that are responsible for anaerobic pigment degradation have not yet been identified, but denitrifying enrichments were obtained on biosynthetic precursors, α- and β-ionone (J. Harder, unpublished). The mineralization of menaquinone and ubiquinone in the absence of molecular oxygen has not been investigated.

5 Cholesterol

The diagenesis of cholesterol has been studied in great detail [43,44]. The most common reaction of several biotransformations which occur on the A ring is a reduction of the carbon-carbon double bond of cholesterol yielding coprostanol [4548]. A mineralization of sterols in anaerobic habitats was determined from lake sediment studies: the total sterol content decreased faster than the total organic carbon pool, but more slowly than the pool of linear long-chain aliphatic alcohols and fatty acids [49]. Recently a denitrifying bacterium, strain 72Chol, was isolated that mineralizes cholesterol in the absence of molecular oxygen completely to carbon dioxide [50].

The hallmark of the cholesterol structure are two quaternary carbon atoms, C-10 and C-13. The degradation of tertiary carbon atoms, e.g. the removal of β-methyl groups, has been investigated but a systematic approach towards quaternary carbon atoms is still lacking. Cytochrome P450 enzymes, estrogen aromatase and lanosterol 14α-demethylase, perform the reduction of three oxygen molecules to dislodge a methyl group as formate from a quaternary carbon atom [51]. Aerobic cholesterol degrading bacteria oxygenate the C-9 atom, and the formed 9α-hydroxy-androsta-1,4-dien-3,17-dione breaks nonenzymatically into 9,10-seco-androsta-1,3,5(10)-trien-3-ol-9,17-dione [3].

The degradation of the C-13 quaternary structure is not known in detail. Schubert et al. [52] prepared [1,3-14C]7a-methyl-5,6,7,7a-tetrahydroindane-1,5-dione-4-(3-propionic acid) and isolated [1,4-14C]succinate as metabolite formed by Nocardia opaca. They concluded that the succinate represents the atoms C-14-C-17 of cholesterol. An alternative explanation, the part C-14-C-13(C-18)-C-17, deserves attention, because methylmalonyl-CoA mutases produces succinyl-CoA and this reaction might be the source of succinate. To address the fate of quaternary carbon atoms, five denitrifying strains were isolated on dimethylmalonate [40]. Future studies may reveal whether the degradation may occur via a dimethylmalonyl CoA mutase or a dimethylmalonyl CoA decarboxylase or an unknown pathway.

The capacity of denitrifying microorganisms to degrade sterols is not limited to cholesterol (5-cholesten-3β-ol). Strain 72Chol grows also on 5α-cholestan-3β-ol [50], cholest-4-en-3-one, 5,7-cholestadien-3β-ol and lithocholic acid [40]. Denitrifying enrichments grew on lithocholic acid, ergosterol, diosgenin, or stigmasterol as sole electron donor (J. Harder, unpublished).

6 Bile acids

Primary bile acids are synthesized from cholesterol in the liver and are conjugated to either glycine or taurine prior to their active secretion from the liver into the gallbladder [53]. In humans, the two primary bile acids are cholic acid (CA) and chenodeoxycholic acid (CDCA) (Fig. 6).

Figure 6

Biosynthesis of bile acids in man. Primary bile acids cholic acid and chenodeoxycholic acid are synthesized in the liver hepatocyte. Cholic acid and chenodeoxycholic acid are biotransformed into deoxycholic acid and lithocholic acid, respectively, by intestinal anaerobic bacteria.

Following secretion, the conjugated bile acids are stored in the gallbladder and released into the small intestine following food intake. In the small intestine, bile acids act as detergents and are required for solubilization, digestion, and absorption of cholesterol, fats, and fat-soluble vitamins. Most bile acids are actively absorbed from the intestines in the terminal ileum and returned to the liver via the portal blood circulation. The cycling of bile acids between the liver, gallbladder, and intestines is referred to as enterohepatic circulation (Fig. 7) and this process is estimated to occur 8–10 times each day [53].

Figure 7

The enterohepatic circulation of bile acids in man.

6.1 Intestinal metabolism of bile acids

Because bile acid absorption from the small intestine is incomplete, several hundred milligrams of bile acids are lost into the colon and as much as 600 mg of bile acids are secreted into the feces each day. In the colon, the bile acids are exposed to approximately 2×1011 to 5×1011 bacteria per gram wet weight of human feces [54]. Up to 400 different kinds of bacteria are found in the colon, and more than 99% are obligate anaerobes. The predominant species include members of the genera Bacteroides, Fusobacterium, Eubacterium, and Clostridium[54].

The intestinal microflora can generate 15–20 different bile acid metabolites from cholic acid and chenodeoxycholic acid [55]. Known biotransformations include deconjugation of primary bile acids at the C-24 position; oxidation of α-hydroxy groups at the C-3, C-7, and C-12 positions, generating various oxo bile acids; and reduction of oxo bile acids to the β-position (Fig. 8). Through the combined action of α- and β-hydroxysteroid dehydrogenases (HSDHs) via an oxo bile acid intermediate, the intestinal microflora can epimerize various hydroxy groups of bile acids. Epimerization can be carried out by a single species of bacteria containing both α- and β-HSDHs (intraspecies), or by protocooperation between two species of bacteria, one containing the α-HSDH, and the second containing the β-HSDH (interspecies). MPN counts of the bacterial population (103–104 culturable cells per gram of feces) indicated that a minor population of Gram-positive anaerobic bacteria can carry out 7α-dehydroxylation of cholic acid and chenodeoxycholic acid, yielding deoxycholic acid and lithocholic acid, respectively (Fig. 6). One-fifth of patients with cholesterol gallstones exhibit increased levels of deoxycholic acid in bile and enlarged 7α-dehydroxylating activities and populations [56]. Ampicillin treatment normalized the bacterial population and relieved the cholesterol supersaturation [56].

Figure 8

Metabolism of conjugated cholic acid by intestinal bacteria. Abbreviations: R-H, glycine or taurine; 7α-HSDH, 7α-hydroxysteroid dehydrogenase; 7β-HSDH, 7β-hydroxysteroid dehydrogenase.

6.2 Cholic acid 7α-dehydroxylation pathway in anaerobic bacteria

Quantitatively, the most important bile acid biotransformation carried out by intestinal bacteria is the 7α-dehydroxylation of primary bile acids. A novel biochemical pathway for bile acid 7α-dehydroxylation has been described for Eubacterium sp. strain VPI 12708, a human intestinal isolate (Fig. 9). Cholic acid is believed to be actively transported into the bacterial cell [57] and ligated to coenzyme A [58]. The cholyl-CoA is then oxidized by a novel 3α- hydroxysteroid dehydrogenase (3α-HSDH) to produce a 3-oxo-cholyl-CoA conjugate [59]. Further oxidation by a bile acid Δ4-steroid oxidoreductase yields a 3-oxo-Δ4-7α,12α-dihydroxy bile acid intermediate. These two oxidation steps may make it chemically easier to remove the 7α-hydroxy group. Dehydration of the 7α-hydroxyl group yields a 3-oxo-Δ4,6-steroid intermediate which is reduced in three subsequent steps, yielding deoxycholic acid. It is unclear when CoA is released from the bile acid intermediate and when, or how, the secondary bile acid is transported out of the bacterial cell (Fig. 9). This pathway recognizes both cholic and chenodeoxycholic acid and may be the major, if not sole, bile acid 7α-dehydroxylation pathway in human intestinal bacteria [60,61]. Similar biochemical reactions appear to be carried out by Clostridium aminovalericum during fermentation of 5-aminovalerate [62]. The key enzyme, 5-hydroxyvaleryl-CoA dehydratase, catalyzes the oxidation of 5-hydroxyvaleryl-CoA to 5-hydroxy-2-pentenoyl-CoA, the dehydration to 2,4-pentadienoyl-CoA and the reduction to 4-pentenoyl-CoA.

Figure 9

Proposed pathway for cholic acid 7α-dehydroxylation in Eubacterium sp. strain VPI 12708. Numbers indicate enzymatic reactions: 1, bile acid transporter (uptake); 2, bile acid coenzyme A ligase; 3, 3α-hydroxysteroid dehydrogenase; 4, 3-oxo-Δ4-steroid oxidoreductase; 5, 7α-dehydratase; 6, Δ6-oxidoreductase; 7, Δ4-oxidoreductase; 8, 3α-hydroxysteroid oxidoreductase; 9, bile acid coenzyme A hydrolase; 10, bile acid transporter (exporter).

6.3 Genetics of bile acid 7α-dehydroxylation in Eubacterium sp. strain VPI 12708

Bile acid 7α-dehydroxylation activity is induced by primary bile acids in Eubacterium sp. strain VPI 12708 [63], as well as other species of Gram-positive anaerobic intestinal bacteria [64]. The induction of 7α-dehydroxylation activity in Eubacterium sp. strain VPI 12708 is associated with the appearance of several new polypeptides as observed with SDS-PAGE analysis. The genes encoding several of these bile acid inducible (bai) polypeptides have been cloned and shown to be part of a large (∼12 kb) operon which encodes nine open reading frames (Fig. 10) [63]. Many of the gene products have been associated with either bile acid transport or specific steps in the cholic acid 7α-dehydroxylation pathway [6567].

Figure 10

Bile acid inducible operon from Eubacteriums sp. strain VPI 12708. Abbreviations: O/P, operator/promoter; 3α-HSDH, 3α-hydroxysteroid dehydrogenase; NADH:FOR, NADH:flavin oxidoreductase.

Interestingly, the Eubacterium sp. strain VPI 12708 genome appears to contain three separate genes encoding 27-kDa bai proteins, which have been called the baiA gene family [68]. Two copies of these genes are identical (baiA1 and baiA3) and encode monocistronic mRNA transcripts [68], and the third gene in the baiA family (baiA2) is one of the genes encoded in the 12-kb bai operon [63]. The baiA2 polypeptide shares approximately 92% amino acid sequence identity with the polypeptides encoded by baiA1 and baiA3. The promoter regions of the large bai operon and baiA1 and baiA3 genes also share considerable (69% over 131 bp) DNA sequence identity, and all three are inducible by cholic acid [63,68].

One additional bai polypeptide associated with the bile acid Δ6- reduction has been isolated from Eubacterium sp. strain VPI 12708. The N-terminal amino acid sequence was determined and this sequence was not encoded by genes found in the large bai operon. Based on this result, additional bile acid inducible genes or operon(s) involved with bile acid 7α-dehydroxylation may be present in this bacterium.

6.4 Enzymology of bile acid 7α-dehydroxylation in Eubacterium sp. strain VPI 12708

Several of the genes encoded by the bai operon have been subcloned and expressed in Escherichia coli[58,59,6567]. Individual genes were amplified by the polymerase chain reaction and cloned into an IPTG-inducible expression vector. Amino acid sequence comparisons were performed in order to determine if these expressed polypeptides were similar to other proteins with known functions.

6.4.1 baiA genes

The baiA genes encode a 27-kDa polypeptide that contains 249 amino acids. The translated amino acid sequence had a significant similarity with the short-chain alcohol/polyol dehydrogenase family [6871] and the bacterial bile acid 7α-hydroxysteroid dehydrogenases [70,71]. When either baiA1 or baiA2 was expressed in E. coli, the purified gene product was found to have bile acid 3α-hydroxysteroid dehydrogenase (3α-HSDH) activity [70]. Surprisingly, the enzymes encoded by these genes recognized only CoA conjugates of bile acids but could utilize either NAD+ or NADP+ as electron acceptors. No catalytic differences were detected between the baiA1 and baiA2 gene products and the physiological significance of multiple genes encoding 3α-HSDH activity in this species is unknown.

6.4.2 baiB gene

The baiB gene encodes a 58-kDa polypeptide containing 521 amino acids. The amino acid sequence shows a high degree of similarity to enzymes that catalyze the ATP-dependent ligation of cyclic carboxylated compounds to AMP or CoA [58]. Based on studies of the baiB expressed in E. coli, the gene appeared to encode a bile acid-CoA ligase activity. The ligation reaction in vitro required unconjugated bile acid, ATP, CoA, and Mg2+ for the synthesis of CoA conjugated bile acids and AMP. The conjugation of primary bile acids to CoA appears to be required prior to the oxidation of the 3α-hydroxyl group by the novel 3α-HSDH (product of the baiA gene family).

6.4.3 baiC and baiH genes

The baiH gene encodes a 72-kDa polypeptide containing 661 amino acids. When this gene was expressed in E. coli, it was found to encode NADH:flavin oxidoreductase activity. Amino acid sequence analysis showed that the baiH gene product is similar to NADH oxidase from Thermoanaerobium brockii, triethylamine dehydrogenase from the methylotropic bacterium W3A1, old yellow enzyme from Saccharomyces carlbergensis, and the baiC gene product from Eubacterium sp. strain VPI 12708, which is a 59.5-kDa polypeptide containing 540 amino acids [66]. The function of the baiC gene is currently unknown, although there is a high degree of DNA and amino acid sequence similarity between the baiH and baiC genes and their products which might suggest that they arose from a common ancestral gene.

The purified NADH:flavin oxidoreductase (baiH) contains 1 mol of FAD, 2 mol of iron, and 1 mol of copper per mol of subunit [67], and reduces quinones, dyes, flavins, and molecular oxygen using NADH as an electron donor. The addition of purified polypeptide to cell extracts of Eubacterium sp. strain VPI 12708 results in a change in the ratio of oxidized to reduced bile acid 7α-dehydroxylation intermediates, suggesting that this enzyme might be involved in the regulation of the cellular NAD+/NADH ratio.

6.4.4 baiE and baiI genes

The baiE gene encodes a 19.5-kDa polypeptide containing 166 amino acids [65]. Expression of the baiE gene in E. coli was associated with a bile acid 7α-dehydratase activity that appeared to exist as a dimer in its active form [65]. The 7α-dehydratase encoded by baiE recognized only 7α,12α-dihydroxy-3-oxo-4-cholenoic acid and 7α-hydroxy-3-oxo-4-cholenoic acid as substrates. Since this enzyme did not use 7β-hydroxy-3-oxo-4-cholenoic acid, it is probably specific for 7α-dehydration of bile acids. It is currently unknown if this enzyme will catalyze 7α-dehydration of bile acid-CoA conjugates. The baiE gene product has significant amino acid similarity to the baiI gene product from Eubacterium sp. strain VPI 12708, but neither share significant homology with any proteins in available databases. No activity has been associated with the baiI gene product.

6.4.5 baiF gene

The baiF gene encodes a 47.5-kDa polypeptide containing 426 amino acids. The amino acid sequence shares significant amino acid similarity (60%) with the E. coli caiB gene product, a 45-kDa carnitine dehydratase. The purified baiF gene product has been shown to have bile acid coenzyme A hydrolase activity. However, no CoA transferase activity or 7α- or 7β-dehydratase activity has been detected [72].

6.4.6 baiG gene

The baiG gene encodes a polypeptide containing 477 amino acids with a significant amino acid sequence homology to several membrane transport proteins, including antibiotic resistance transporters which are members of the major facilitator superfamily. The highest degree of similarity was found with class K and L TetA proteins from Gram-positive bacteria. Based on computer membrane protein modeling, the baiG polypeptide should have 14 transmembrane domains [57]. When the baiG gene was expressed in E. coli, the polypeptide product conferred H+-dependent bile acid transport activity that preferred cholic acid and chenodeoxycholic acid. Secondary bile acids and conjugated bile acids were transported to a much lesser degree.

7 Conclusions

A small population of intestinal anaerobic bacteria 7α-dehydroxylates primary bile acids using a six-step biochemical pathway. This pathway appears to serve as an electron sink during fermentative metabolism by these organisms. Several enzymes catalyzing specific reactions in this pathway have been characterized and shown to belong to novel gene families.

The isolation of denitrifying bacteria on monoterpenes and cholesterol proves the involvement of respiring anaerobes in the mineralization of isoprenoic compounds in nature. The organisms present a new source for novel biotransformations and unusual enzyme activities. Future studies are required to understand the anaerobic mineralization pathways of natural substances. This will benefit the assessment of the degradability of man-made compounds in anaerobic ecosystems, similar to the relatedness of degradative pathways for natural and man-made substances in aerobic microorganisms [73,74].


This work was supported by Grant PO1 DK38030 to P.B.H. The Max Planck Society and grants from the Deutsche Forschungsgemeinschaft supported the research of J.H.


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View Abstract