Biochemical and genetic characterization of benzylsuccinate synthase from <i>Thauera aromatica</i>: a new glycyl radical enzyme catalysing the first step in anaerobic toluene metabolism

Leuthner, Birgitta; Leutwein, Christina; Schulz, Henk; Hörth, Patric; Haehnel, Wolfgang; Schiltz, Emile; Heider, Johann

doi:10.1046/j.1365-2958.1998.00826.x

Cited by 284 publications

(372 citation statements)

References 37 publications

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“…The specific activity of the cell extract at 4 °C was 2.4 nmol/min/mg of protein, whereas at 30 °C the specific activity rose to 7.4 nmol/min/mg. This level of activity is similar to that reported in permeablized cells of Azoracus sp (18), and some 4 orders of magnitude higher than that reported for purified enzyme from T. aromatica (6). As judged by SDS-PAGE, the BSS α-subunit, a conspicuous band of M r 98,000, comprised about 3.5 % of the total proteins present in the cell free extract, which allows us to estimate k cat for BSS as approximately 0.11 s −1 at 4 °C and 0.35 s −1 at 30°C .…”

Section: Enzyme Assaysupporting

confidence: 88%

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Deuterium Isotope Effects in the Unusual Addition of Toluene to Fumarate Catalyzed by Benzylsuccinate Synthase

Marsh

2006

Biochemistry

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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 that is catalyzed by benzylsuccinate synthase. Benzylsuccinate synthase is a member of the glycyl radicalcontaining 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 V max and V max /K m when deuterated toluene is the substrate. At 30 °C the isotope effects are 1.7 ± 0.2 and 2.9 ± 0.1 on V max and V max /K m , respectively, at 4 °C they increase slightly to2.2 ± 0.2 and 3.1 ± 0.1 respectively. We compare these results with the theoretical isotope effects on V max and V max /K m that are predicted from the free energy profile for the uncatalyzed reaction, which has previously been computed using density functional theory. The comparison allows us to draw some conclusions on how the enzyme may catalyze this unusual reaction.Keywords toluene metabolism; free radicals; enzyme kinetics; Tauera. aromatica; glycyl radical enzyme Toluene and related aromatic compounds represent an important class of pollutants that are long-lived in the environment and have relatively high water solubility. This is a particular problem in environments where oxygen is scarce, such as ground water supplies. It was initially thought that the only routes to degrade such compounds were through oxidative pathways that require molecular oxygen to functionalize otherwise inert aromatic hydrocarbons. However, the recent discovery in bacteria of anaerobic pathways for the degradation of aromatic compounds has aroused much interest, both for the potential they offer for bio-remediation in situations where oxygen is scarce, and because of the novel chemistry involved (1,2).Various denitrifying and sulfate-reducing bacteria such as Thauera aromatica and Desulfobacula toluolica are able to live on toluene as their sole source of carbon and energy under anaerobic conditions (3,4). The first step in this pathway is the addition of toluene across the double bond of fumarate to produce (R)-benzylsuccinate, as shown in scheme 1, in which hydrogen in the methyl group of toluene is retained in the product (5). This remarkable chemical transformation is catalyzed by benzylsuccinate synthase 1 (BSS). † This research was supported by a grant from the National Institutes of Health, GM 59227 to E.N.G.M. BSS as a member of the growing class of glycyl radical-containing enzymes that includes pyruvate formate-lyase (PFL) and anaerobic ribonucleotide reductase (6-11). EPR studies show that the resting enzyme harbors an organic radical similar to that observed in these enzymes (12)(13)(14). Like other glycyl radical enzymes, BSS is extremely oxygen sensitive, and exposure to air results in oxidative cleavage of the α subunit at the site of the presumed glycyl radical (6).We recently investigated the stereochemistr...

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Section: Enzyme Assaysupporting

confidence: 88%

“…EPR studies show that the resting enzyme harbors an organic radical similar to that observed in these enzymes (12)(13)(14). Like other glycyl radical enzymes, BSS is extremely oxygen sensitive, and exposure to air results in oxidative cleavage of the α subunit at the site of the presumed glycyl radical (6).…”

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confidence: 68%

Deuterium Isotope Effects in the Unusual Addition of Toluene to Fumarate Catalyzed by Benzylsuccinate Synthase

Marsh

2006

Biochemistry

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“…The lowest level of identity (around 25%) was found with the well characterized PFLs PflB from E. coli (28) and C. pasteurianum (29). The highest degree of identity was obtained with PflD and f810 (41% and 37%, respectively), two Pfl homologues from E. coli with unknown functions (30), and the recently characterized enzyme involved in anaerobic toluene metabolism, benzylsuccinate synthase from Thauera aromatica (32% identity) (31) and TutD from Thauera sp. T1 (30% identity) (32).…”

Section: Resultsmentioning

confidence: 99%

Molecular characterization of the 1,3-propanediol (1,3-PD) operon of Clostridium butyricum

Raynaud

Sarçabal

Meynial-Salles

et al. 2003

Proc. Natl. Acad. Sci. U.S.A.

207

160

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The genes encoding the 1,3-propanediol (1,3-PD) operon of Clostridium butyricum VPI1718 were characterized from a molecular and a biochemical point of view. This operon is composed of three genes, dhaB1, dhaB2, and dhaT. When grown in a vitamin B12-free mineral medium with glycerol as carbon source, Escherichia coli expressing dhaB1, dhaB2, and dhaT produces 1,3-PD and high glycerol dehydratase and 1,3-PD dehydrogenase activities. dhaB1 and dhaB2 encode, respectively, a new type of glycerol dehydratase and its activator protein. The deduced proteins DhaB1 and DhaB2, with calculated molecular masses of 88,074 and 34,149 Da, respectively, showed no homology with the known glycerol dehydratases that are all B12 dependent but significant similarity with the pyruvate formate lyases and pyruvate formate lyases activating enzymes and their homologues. The 1,158-bp dhaT gene codes for a 1,3-PD dehydrogenase with a calculated molecular mass of 41,558 Da, revealing a high level of identity with other DhaT proteins from natural 1,3-PD producers. The expression of the 1,3-PD operon in C. butyricum is regulated at the transcriptional level, and this regulation seems to involve a two-component signal transduction system DhaAS͞DhaA, which may have a similar function to DhaR, a transcriptional regulator found in other natural 1,3-PD producers. The discovery of a glycerol dehydratase, coenzyme B12 independent, should significantly influence the development of an economical vitamin B12-free biological process for the production of 1,3-PD from renewable resources.

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“…Because of their oxygen-sensitive nature, GREs (11)(12)(13)(14) are proposed to have evolved before the appearance of oxygen in the atmosphere, and some speculate that class III RNR may be a truly ancient enzyme (15) because the ancestral RNR was likely involved in the conversion of RNA-to DNA-based life (16). Unlike RNR, which converts ribonucleotides to deoxyribonucleotides, PFL is a central metabolic enzyme that converts pyruvate and CoA to acetyl-CoA and formate (17).…”

mentioning

confidence: 99%

Structural basis for glycyl radical formation by pyruvate formate-lyase activating enzyme

Vey

Yang²,

Li³

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

174

280

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Pyruvate formate-lyase activating enzyme generates a stable and catalytically essential glycyl radical on G 734 of pyruvate formatelyase via the direct, stereospecific abstraction of a hydrogen atom from pyruvate formate-lyase. The activase performs this remarkable feat by using an iron-sulfur cluster and S-adenosylmethionine (AdoMet), thus placing it among the AdoMet radical superfamily of enzymes. We report here structures of the substrate-free and substrate-bound forms of pyruvate formate-lyase-activating enzyme, the first structures of an AdoMet radical activase. To obtain the substrate-bound structure, we have used a peptide substrate, the 7-mer RVSGYAV, which contains the sequence surrounding G 734 . Our structures provide fundamental insights into the interactions between the activase and the G 734 loop of pyruvate formate-lyase and provide a structural basis for direct and stereospecific H atom abstraction from the buried G 734 of pyruvate formate-lyase.crystallography ͉ metalloprotein ͉ radical chemistry ͉ S-adenosylmethionine ͉ iron-sulfur cluster

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Biochemical and genetic characterization of benzylsuccinate synthase from Thauera aromatica: a new glycyl radical enzyme catalysing the first step in anaerobic toluene metabolism

Cited by 284 publications

References 37 publications

Deuterium Isotope Effects in the Unusual Addition of Toluene to Fumarate Catalyzed by Benzylsuccinate Synthase

Deuterium Isotope Effects in the Unusual Addition of Toluene to Fumarate Catalyzed by Benzylsuccinate Synthase

Molecular characterization of the 1,3-propanediol (1,3-PD) operon of Clostridium butyricum

Structural basis for glycyl radical formation by pyruvate formate-lyase activating enzyme

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