Analysis of the Novel Benzylsuccinate Synthase Reaction for Anaerobic Toluene Activation Based on Structural Studies of the Product

Recent studies of anaerobic toluene catabolism have demonstrated a novel reaction for anaerobic hydrocarbon activation: the addition of the methyl carbon of toluene to fumarate to form benzylsuccinate. In vitro studies of the anaerobic benzylsuccinate synthase reaction indicate that the H atom abstracted from the toluene methyl group during addition to fumarate is retained in the succinyl moiety of benzylsuccinate. Based on structural studies of benzylsuccinate formed during anaerobic, in vitro assays with denitrifying, toluene-mineralizing strain T, we now report the following characteristics of the benzylsuccinate synthase reaction: (i) it is highly stereospecific, resulting in >95% formation of the (+)-benzylsuccinic acid enantiomer [(R)-2-benzyl-3-carboxypropionic acid], and (ii) active benzylsuccinate synthase does not contain an abstracted methyl H atom from toluene at the beginning or at the end of a catalytic cycle.     

New glycyl radical enzymes catalysing key metabolic steps in anaerobic bacteria

During the last decade, an increasing number of new enzymes containing glycyl radicals in their active sites have been identified and biochemically characterised. These include benzylsuccinate synthase (Bss), 4-hydroxyphenylacetate decarboxylase (Hpd) and the coenzyme B12-independent glycerol dehydratase (Gdh). These are involved in metabolic pathways as different as anaerobic toluene metabolism, fermentative production of p-cresol and glycerol fermentation. Some features of these newly discovered enzymes are described and compared with those of the previously known glycyl radical enzymes pyruvate formate-lyase (Pfl) and anaerobic ribonucleotide reductase (Nrd). Among the new enzymes, Bss and Hpd share the presence of small subunits, the function of which in the catalytic mechanisms is still enigmatic, and both enzymes contain metal centres in addition to the glycyl radical prosthetic group. The activating enzymes of the novel systems also deviate from the standard type, containing at least one additional Fe-S cluster. Finally, the available whole-genome sequences of an increasing number of strictly or facultative anaerobic bacteria revealed the presence of many more hitherto unknown glycyl radical enzyme (GRE) systems. Recent studies suggest that the particular types of these enzymes represent the ends of different evolutionary lines, which emerged early in evolution and diversified to yield remarkably versatile biocatalysts for chemical reactions that are otherwise difficult to perform in anoxic environments.     

Stable Hydrogen and Carbon Isotope Fractionation during Microbial Toluene Degradation: Mechanistic and Environmental Aspects

Primary features of hydrogen and carbon isotope fractionation during toluene degradation were studied to evaluate if analysis of isotope signatures can be used as a tool to monitor biodegradation in contaminated aquifers. D/H hydrogen isotope fractionation during microbial degradation of toluene was measured by gas chromatography. Per-deuterated toluene-d₈ and nonlabeled toluene were supplied in equal amounts as growth substrates, and kinetic isotope fractionation was calculated from the shift of the molar ratios of toluene-d₈ and nondeuterated toluene. The D/H isotope fractionation varied slightly for sulfate-reducing strain TRM1 (slope of curve [b] = −1.219), Desulfobacterium cetonicum (b = −1.196), Thauera aromatica (b = −0.816), and Geobacter metallireducens (b = −1.004) and was greater for the aerobic bacterium Pseudomonas putida mt-2 (b = −2.667). The D/H isotope fractionation was 3 orders of magnitude greater than the ¹³C/¹²C carbon isotope fractionation reported previously. Hydrogen isotope fractionation with nonlabeled toluene was 1.7 and 6 times less than isotope fractionation with per-deuterated toluene-d₈ and nonlabeled toluene for sulfate-reducing strain TRM1 (b = −0.728) and D. cetonicum (b = −0.198), respectively. Carbon and hydrogen isotope fractionation during toluene degradation by D. cetonicum remained constant over a growth temperature range of 15 to 37°C but varied slightly during degradation by P. putida mt-2, which showed maximum hydrogen isotope fractionation at 20°C (b = −4.086) and minimum fractionation at 35°C (b = −2.138). D/H isotope fractionation was observed only if the deuterium label was located at the methyl group of the toluene molecule which is the site of the initial enzymatic attack on the substrate by the bacterial strains investigated in this study. Use of ring-labeled toluene-d₅ in combination with nondeuterated toluene did not lead to significant D/H isotope fractionation. The activity of the first enzyme in the anaerobic toluene degradation pathway, benzylsuccinate synthase, was measured in cell extracts of D. cetonicum with an initial activity of 3.63 mU (mg of protein)⁻¹. The D/H isotope fractionation (b = −1.580) was 30% greater than that in growth experiments with D. cetonicum. Mass spectroscopic analysis of the product benzylsuccinate showed that H atoms abstracted from the toluene molecules by the enzyme were retained in the same molecules after the product was released. Our findings revealed that the use of deuterium-labeled toluene was appropriate for studying basic features of D/H isotope fractionation. Similar D/H fractionation factors for toluene degradation by anaerobic bacteria, the lack of significant temperature dependence, and the strong fractionation suggest that analysis of D/H fractionation can be used as a sensitive tool to assess degradation activities. Identification of the first enzyme reaction in the pathway as the major fractionating step provides a basis for linking observed isotope fractionation to biochemical reactions.     

Stable hydrogen and carbon isotope fractionation during microbial toluene degradation: mechanistic and environmental aspects

Primary features of hydrogen and carbon isotope fractionation during toluene degradation were studied to evaluate if analysis of isotope signatures can be used as a tool to monitor biodegradation in contaminated aquifers. D/H hydrogen isotope fractionation during microbial degradation of toluene was measured by gas chromatography. Per-deuterated toluene-d(8) and nonlabeled toluene were supplied in equal amounts as growth substrates, and kinetic isotope fractionation was calculated from the shift of the molar ratios of toluene-d(8) and nondeuterated toluene. The D/H isotope fractionation varied slightly for sulfate-reducing strain TRM1 (slope of curve [b] = -1.219), Desulfobacterium cetonicum (b = -1.196), Thauera aromatica (b = -0.816), and Geobacter metallireducens (b = -1.004) and was greater for the aerobic bacterium Pseudomonas putida mt-2 (b = -2.667). The D/H isotope fractionation was 3 orders of magnitude greater than the (13)C/(12)C carbon isotope fractionation reported previously. Hydrogen isotope fractionation with nonlabeled toluene was 1.7 and 6 times less than isotope fractionation with per-deuterated toluene-d(8) and nonlabeled toluene for sulfate-reducing strain TRM1 (b = -0.728) and D. cetonicum (b = -0.198), respectively. Carbon and hydrogen isotope fractionation during toluene degradation by D. cetonicum remained constant over a growth temperature range of 15 to 37 degrees C but varied slightly during degradation by P. putida mt-2, which showed maximum hydrogen isotope fractionation at 20 degrees C (b = -4.086) and minimum fractionation at 35 degrees C (b = -2.138). D/H isotope fractionation was observed only if the deuterium label was located at the methyl group of the toluene molecule which is the site of the initial enzymatic attack on the substrate by the bacterial strains investigated in this study. Use of ring-labeled toluene-d(5) in combination with nondeuterated toluene did not lead to significant D/H isotope fractionation. The activity of the first enzyme in the anaerobic toluene degradation pathway, benzylsuccinate synthase, was measured in cell extracts of D. cetonicum with an initial activity of 3.63 mU (mg of protein)(-1). The D/H isotope fractionation (b = -1.580) was 30% greater than that in growth experiments with D. cetonicum. Mass spectroscopic analysis of the product benzylsuccinate showed that H atoms abstracted from the toluene molecules by the enzyme were retained in the same molecules after the product was released. Our findings revealed that the use of deuterium-labeled toluene was appropriate for studying basic features of D/H isotope fractionation. Similar D/H fractionation factors for toluene degradation by anaerobic bacteria, the lack of significant temperature dependence, and the strong fractionation suggest that analysis of D/H fractionation can be used as a sensitive tool to assess degradation activities. Identification of the first enzyme reaction in the pathway as the major fractionating step provides a basis for linking observed isotope fractionation to biochemical reactions.     

Anaerobic Toluene Activation by Benzylsuccinate Synthase in a Highly Enriched Methanogenic Culture

Permeabilized cells of a highly enriched, toluene-mineralizing, methanogenic culture catalyzed the addition of toluene to fumarate to form benzylsuccinate under anaerobic conditions. The specific in vitro rate of benzylsuccinate formation was >85% of the specific in vivo rate of toluene consumption. This is the first report of benzylsuccinate synthase activity in a methanogenic culture; the activity has previously been reported to occur in denitrifying, sulfate-reducing, and anoxygenic phototrophic bacteria.     

6-Oxocyclohex-1-ene-1-carbonyl-coenzyme A hydrolases from obligately anaerobic bacteria: characterization and identification of its gene as a functional marker for aromatic compounds degrading anaerobes

In anaerobic bacteria, most aromatic growth substrates are channelled into the benzoyl-coenzyme A (CoA) degradation pathway where the aromatic ring is dearomatized and cleaved into an aliphatic thiol ester. The initial step of this pathway is catalysed by dearomatizing benzoyl-CoA reductases yielding the two electron-reduction product, cyclohexa-1,5-diene-1-carbonyl-CoA, to which water is subsequently added by a hydratase. The next two steps have so far only been studied in facultative anaerobes and comprise the oxidation of the 6-hydroxyl-group to 6-oxocyclohex-1-ene-1-carbonyl-CoA (6-OCH-CoA), the addition of water and hydrolytic ring cleavage yielding 3-hydroxypimelyl-CoA. In this work, two benzoate-induced genes from the obligately anaerobic bacteria, Geobacter metallireducens (bamA(Geo)) and Syntrophus aciditrophicus (bamA(Syn)), were heterologously expressed in Escherichia coli, purified and characterized as 6-OCH-CoA hydrolases. Both enzymes consisted of a single 43 kDa subunit. Some properties of the enzymes are presented and compared with homologues from facultative anaerobes. An alignment of the nucleotide sequences of bamA(Geo) and bamA(Syn) with the corresponding genes from facultative anaerobes identified highly conserved DNA regions, which enabled the discrimination of genes coding for 6-OCH-CoA hydrolases from those coding for related enzymes. A degenerate oligonucleotide primer pair was deduced from conserved regions and applied in polymerase chain reaction reactions. Using these primers, the expected DNA fragment of the 6-OCH-CoA hydrolase genes was specifically amplified from the DNA of nearly all known facultative and obligate anaerobes that use aromatic growth substrates. The only exception was the aromatic compound-degrading Rhodopseudomonas palustris, which uniquely uses a modified benzoyl-CoA degradation pathway. Using the oligonucleotide primers, the expected DNA fragment was also amplified in a toluene-degrading and a m-xylene-degrading enrichment culture demonstrating its potential use in less defined bacterial communities. The gene probe established in this work provides for the first time a general tool for the detection of a central functionality in aromatic compound-degrading anaerobes.     

A stable organic free radical in anaerobic benzylsuccinate synthase of Azoarcus sp. strain T

The novel enzyme benzylsuccinate synthase initiates anaerobic toluene metabolism by catalyzing the addition of toluene to fumarate, forming benzylsuccinate. Based primarily on its sequence similarity to the glycyl radical enzymes, pyruvate formate-lyase and anaerobic ribonucleotide reductase, benzylsuccinate synthase was speculated to be a glycyl radical enzyme. In this report we use EPR spectroscopy to demonstrate for the first time that active benzylsuccinate synthase from the denitrifying bacterium Azoarcus sp. strain T harbors an oxygen-sensitive stable organic free radical. The EPR signal of the radical was centered at g = 2.0021 and was characterized by a major 2-fold splitting of about 1.5 millitesla. The strong similarities between the EPR signal of the benzylsuccinate synthase radical and that of the glycyl radicals of pyruvate formate-lyase and anaerobic ribonucleotide reductase provide evidence that the benzylsuccinate synthase radical is located on a glycine residue, presumably glycine 828 in Azoarcus sp. strain T benzylsuccinate synthase.     

Cyclohexa-1,5-diene-1-carbonyl-coenzyme A (CoA) hydratases of Geobacter metallireducens and Syntrophus aciditrophicus: Evidence for a common benzoyl-CoA degradation pathway in facultative and strict anaerobes

In the denitrifying bacterium Thauera aromatica, the central intermediate of anaerobic aromatic metabolism, benzoyl-coenzyme A (CoA), is dearomatized by the ATP-dependent benzoyl-CoA reductase to cyclohexa-1,5-diene-1-carbonyl-CoA (dienoyl-CoA). The dienoyl-CoA is further metabolized by a series of beta-oxidation-like reactions of the so-called benzoyl-CoA degradation pathway resulting in ring cleavage. Recently, evidence was obtained that obligately anaerobic bacteria that use aromatic growth substrates do not contain an ATP-dependent benzoyl-CoA reductase. In these bacteria, the reactions involved in dearomatization and cleavage of the aromatic ring have not been shown, so far. In this work, a characteristic enzymatic step of the benzoyl-CoA pathway in obligate anaerobes was demonstrated and characterized. Dienoyl-CoA hydratase activities were determined in extracts of Geobacter metallireducens (iron reducing), Syntrophus aciditrophicus (fermenting), and Desulfococcus multivorans (sulfate reducing) cells grown with benzoate. The benzoate-induced genes putatively coding for the dienoyl-CoA hydratases in the benzoate degraders G. metallireducens and S. aciditrophicus were heterologously expressed and characterized. Both gene products specifically catalyzed the reversible hydration of dienoyl-CoA to 6-hydroxycyclohexenoyl-CoA (Km, 80 and 35 microM; Vmax, 350 and 550 micromol min(-1) mg(-1), respectively). Neither enzyme had significant activity with cyclohex-1-ene-1-carbonyl-CoA or crotonyl-CoA. The results suggest that benzoyl-CoA degradation proceeds via dienoyl-CoA and 6-hydroxycyclohexanoyl-CoA in strictly anaerobic bacteria. The steps involved in dienoyl-CoA metabolism appear identical in all nonphotosynthetic anaerobic bacteria, although totally different benzene ring-dearomatizing enzymes are present in facultative and obligate anaerobes.     

感染根管的厌氧菌培养结果分析

从64只感染根管中的58只根管分离到144株无芽胞厌氧菌,其中类杆菌54株,厌氧性链球菌23株,韦荣氏球菌17株,真杆菌11株,梭杆菌10株,放线菌8株,双岐杆菌2株,消化链球菌和消化球菌19株。40只根管为厌氧菌和兼性厌氧菌或需氧菌混合感染,18只根管和6只根管分别为单独厌氧菌和兼性厌氧菌感染。33只根尖周炎根管分别采集牙髓和根尖渗出物样本进行培养,实验结果表明牙髓样本中革兰氏阳性厌氧杆菌检出率较高,根尖渗出物中以产黑素类杆菌属的细菌检出率较高。根尖周炎和牙槽脓肿患者的感染根管中产黑素类杆菌属的细菌检出率明显高于蜂窝组织炎患者。     

Anaerobic toluene activation by benzylsuccinate synthase in a highly enriched methanogenic culture

Permeabilized cells of a highly enriched, toluene-mineralizing, methanogenic culture catalyzed the addition of toluene to fumarate to form benzylsuccinate under anaerobic conditions. The specific in vitro rate of benzylsuccinate formation was >85% of the specific in vivo rate of toluene consumption. This is the first report of benzylsuccinate synthase activity in a methanogenic culture; the activity has previously been reported to occur in denitrifying, sulfate-reducing, and anoxygenic phototrophic bacteria.     

Deuterium isotope effects in the unusual addition of toluene to fumarate catalyzed by benzylsuccinate synthase

The first step in the anaerobic metabolism of toluene is a highly unusual reaction: the addition of toluene across the double bond of fumarate to produce (R)-benzylsuccinate, which is catalyzed by benzylsuccinate synthase. Benzylsuccinate synthase is a member of the glycyl radical-containing family of enzymes, and the reaction is initiated by abstraction of a hydrogen atom from the methyl group of toluene. To gain insight into the free energy profile of this reaction, we have measured the kinetic isotope effects on Vmax and Vmax/Km when deuterated toluene is the substrate. At 30 degrees C the isotope effects are 1.7 +/- 0.2 and 2.9 +/- 0.1 on Vmax and Vmax/Km, respectively; at 4 degrees C they increase slightly to 2.2 +/- 0.2 and 3.1 +/- 0.1, respectively. We compare these results with the theoretical isotope effects on Vmax and Vmax/Km that are predicted from the free energy profile for the uncatalyzed reaction, which has previously been computed using density functional theory [Himo, F. (2002) J. Phys. Chem. B 106, 7688-7692]. The comparison allows us to draw some conclusions on how the enzyme may catalyze this unusual reaction.     

How obligatory is anaerobiosis?

Historically many bacteria have been classified as obligate anaerobes. They have been construed as wholly intolerant of oxygen, a feature that was originally ascribed to their lack of superoxide dismutases and catalases. Clostridial species were regarded as classic examples. We now know that this view is quite wrong: enzymes that scavenge superoxide, hydrogen peroxide and even oxygen itself abound in anaerobes. In the current issue of Molecular Microbiology, Hillmann et al. demonstrate that full production of these proteins can allow Clostridium acetobutylicum to survive and even grow in oxygenated culture medium. Evidently, oxidative defences in anaerobes can be robust. In all likelihood, they are critical for the movement of bacteria through aerobic environments to new anaerobic habitats.     

Substrate-bound Structures of Benzylsuccinate Synthase Reveal How Toluene Is Activated in Anaerobic Hydrocarbon Degradation

Various bacteria perform anaerobic degradation of small hydrocarbons as a source of energy and cellular carbon. To activate non-reactive hydrocarbons such as toluene, enzymes conjugate these molecules to fumarate in a radical-catalyzed, C—C bond-forming reaction. We have determined x-ray crystal structures of the glycyl radical enzyme that catalyzes the addition of toluene to fumarate, benzylsuccinate synthase (BSS), in two oligomeric states with fumarate alone or with both substrates. We find that fumarate is secured at the bottom of a long active site cavity with toluene bound directly above it. The two substrates adopt orientations that appear ideal for radical-mediated C—C bond formation; the methyl group of toluene is positioned between fumarate and a cysteine that forms a thiyl radical during catalysis, which is in turn adjacent to the glycine that serves as a radical storage residue. Toluene is held in place by fumarate on one face and tight packing by hydrophobic residues on the other face and sides. These hydrophobic residues appear to become ordered, thus encapsulating toluene, only in the presence of BSSβ, a small protein subunit that forms a tight complex with BSSα, the catalytic subunit. Enzymes related to BSS are able to metabolize a wide range of hydrocarbons through attachment to fumarate. Using our structures as a guide, we have constructed homology models of several of these “X-succinate synthases” and determined conservation patterns that will be useful in understanding the basis for catalysis and specificity in this family of enzymes.     

Co-metabolic conversion of toluene in anaerobic n-alkane-degrading bacteria

Diverse microorganisms have been described to degrade petroleum hydrocarbons anaerobically. Strains able to utilize n-alkanes do not grow with aromatic hydrocarbons, whereas strains able to utilize aromatic hydrocarbons do not grow with n-alkanes. To investigate this specificity in more detail, three anaerobic n-alkane degraders (two denitrifying, one sulfate-reducing) and eight anaerobic alkylbenzene degraders (five denitrifying, three sulfate-reducing) were incubated with mixtures of n-alkanes and toluene. Whereas the toluene degradationers formed only the characteristic toluene-derived benzylsuccinate and benzoate, but no n-alkane-derived metabolites, the n-alkane degraders formed toluene-derived benzylsuccinate, 4-phenylbutanoate, phenylacetate and benzoate besides the regular n-alkane-derived (1-methylalkyl)succinates and methyl-branched alkanoates. The co-metabolic conversion of toluene by anaerobic n-alkane degraders to the level of benzoate obviously follows the anaerobic n-alkane degradation pathway with C-skeleton rearrangement and decarboxylation rather than the β-oxidation pathway of anaerobic toluene metabolism. Hence, petroleum-derived aromatic metabolites detectable in anoxic environments may not be exclusively formed by genuine alkylbenzene degraders. In addition, the hitherto largely unexplored fate of fumarate hydrogen during the activation reactions was examined with (2,3-(2) H(2) )fumarate as co-substrate. Deuterium was completely exchanged with hydrogen at the substituted carbon atom (C-2) of the succinate adducts of n-alkanes, whereas it is retained in toluene-derived benzylsuccinate, regardless of the type of enzyme catalysing the fumarate addition reaction.     

Dysbiosis in inflammatory bowel diseases: the oxygen hypothesis

The healthy intestine is characterized by a low level of oxygen and by the presence of large bacterial communities of obligate anaerobes. Dysbiosis of the gut microbiota has been reported in patients suffering from inflammatory bowel diseases (IBDs), but the mechanisms causing this imbalance remain unknown. Observations have included a decrease in obligate anaerobes of the phylum Firmicutes and an increase in facultative anaerobes, including members of the family Enterobacteriaceae. The shift of bacterial communities from obligate to facultative anaerobes strongly suggests a disruption in anaerobiosis and points to a role for oxygen in intestinal dysbiosis. Proposals to evaluate this hypothesis of a role for oxygen in IBD dysbiosis are provided. If this hypothesis is confirmed, decreasing oxygen in the intestine could open novel means to rebalance the microbiota and could provide novel preventative or therapeutic strategies for IBD patients in whom current treatments are ineffective.     

Oral anaerobes cannot survive oxygen stress without interacting with facuItative/aerobic species as a microbial commmunity

D.J. Bradshaw, P.D. Marsh, G.K. Watson and C. Allison. 1997. Anaerobic bacteria are found commonly as components of mixed culture biofilms in many aerated habitats, including the mouth. Previous studies showed that anaerobes could survive in planktonic and biofilm communities in aerated conditions when part of a community including facultative and/or aerobic species, and the numbers and proportions of anaerobic species increased as biofilms aged. When the obligate anaerobes were grown in the absence of aerobic/facultative species, however, they were unable to grow in either the planktonic or biofilm culture. The mean survival times of organisms in the aerated culture containing four anaerobic species varied from around 5 min for Fusobacterium nucleatum and Veillonella dispar , to less than 4 min for Porphyromonas gingivalis and Prevotella nigrescens . In addition, in this culture, the biofilm mode of growth did not provide a haven for these bacteria in the absence of oxygen-consuming species.     

Simple Method for Culturing Anaerobes

A simple, effective method is needed for growing obligate anaerobes in the clinical laboratory. This report describes a pre-reduced anaerobic bottle that can be taken to the bedside for direct inoculation, provides a flat agar surface for evaluation of number and morphology of colonies, and can be incubated in conventional bacteriological incubators. Each anaerobic culture set consisted of two bottles containing brain heart infusion agar and CO₂. Gentamicin sulfate (50 μg/ml) was added to one of these to inhibit facultative enteric bacilli. Comparison of the anaerobic bottles with an identical aerobic bottle which was also routinely inoculated permitted early identification of anaerobic colonies. Representative species of most anaerobic genera of proven pathogenicity for man have been isolated from this system during 10 months of routine use.     

Characteristics of bacteria isolated by the anaerobic roll-tube method from cheeses and ground beef.

In this study the methods of Hungate were used to quantitate the anaerobic bacteria present in commercially available ground beef, cheddar cheese, and German hand cheese. Of 235 anaerobic roll-tube isolates from ground beef and German hand cheese, all were facultative anaerobes. Of 213 anaerobic roll-tube isolates from cheddar cheese, 91% were facultative anaerobes and 9% were obligate anaerobes. Using results of biochemical tests, 14 or the 17 obligately anaerobic isolates from cheddar cheese were Propionibacterium acnes, two were strains of Propionibacterium that could not be speciated, and one was tentatively identified as a strain of Streptococcus evolutus. Obligate anaerobes were estimated to be present in the cheddar cheese at a level of about 10(6)/g. The possible significance of these levels of P. acnes in nonsterile foods is discussed.     

Effect of antibiotics on the human intestinal flora in mice

Antibiotics used during selective decontamination were studied for their effect on the human intestinal flora in mice. Polymyxin B and neomycin were found to eliminate Escherichia coli from the gastrointestinal tract but did not alter total numbers of obligate anaerobes. Neomycin induced an increase of the percentage of gram-negative obligate anaerobes. Cephradine did not affect the numbers of obligate and facultative anaerobes but increased the percentage of gram-negative obligately anaerobic rods in the flora. The selective effect of polymyxin B and neomycin on the flora is accounted for by a relative insusceptibility of the anaerobic flora as compared with E. coli. Low concentrations of polymyxin B and neomycin were detected in caecal supernatants. This was found to be due to strong binding of both antibiotics to the solid fraction of intestinal contents.