Biochemistry of anaerobic biodegradation of aromatic compounds

Fuchs, Georg; Mohamed, Magdy; Altenschmidt, Uwe; Koch, Juergen; Lack, Achim; Brackmann, Ruth; Lochmeyer, Christa; Oswald, B

doi:10.1007/978-94-011-1687-9_16

Cited by 91 publications

(70 citation statements)

References 209 publications

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“…The present data, together with information from previous work (Fuchs et al, 1994), indicate that ABzCoA-M/R discloses a novel pathway located between aerobic and anaerobic catabolic pathways of anthranilate catabolism by facultative anaerobic Pseudornonas strains. In this a novel type of mechanism appears to be operative in which the resonance energy of the aromatic substrate is overwhelmed by a combination of oxygenation and hydrogenation.…”

Section: Mechanistic Conclusionsupporting

confidence: 78%

Kinetic and Mechanistic Studies on the Reactions of 2‐Aminobenzoyl‐CoA Monooxygenase/Reductase

Langkau

Ghisla

1995

European Journal of Biochemistry

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The kinetic mechanism of the flavoprotein 2-aminobenzoyl-CoA monooxygenase lreductase with its natural substrates 2-aminobenzoyl-CoA, NADH and 0, has been investigated using the stopped-flow technique. Initial rate measurements indicate the formation of a ternary complex between oxidized enzyme and the two substrates 2-aminobenzoyl-CoA and NADH, a turnover number of =40 m i x ' was found at pH 7.4 and 4°C. 2-Aminobenzoyl-CoA binds to oxidized enzyme to form a complex which is in a =1: 1 equilibrium with a second, spectrophotometrically distinguishable one. Binding of 2-amino benzoyl-CoA to reduced enzyme is, in contrast, a simple second-order process. Reduction of oxidized enzyme, both uncomplexed and in complex with 2-aminobenzoyl-CoA, by NADH is strongly biphasic. The first fast phase yields enzyme in which 50% of the total FAD is reduced to the FADH, state. This rate is not affected by the presence of 2-aminobenzoyl-CoA. In contrast, 2-aminobenzoyl-CoA enhances -100-fold the second phase, the reduction of the residual 50% FAD. This second phase of reduction (kobs = 2.0 s-') is partially rate-limiting in catalysis. The oxygen reaction of uncomplexed, reduced enzyme is also biphasic and no oxygenated intermediate was detected. Reoxidation of substrate-complexed, reduced enzyme involves three spectroscopically distinguishable species. The first observable intermediate is highly fluorescent suggesting that it consists largely of flavin-4a-hydroxide. Thus, insertion of oxygen into 2-aminobenzoyl-CoA is essentially complete at this point and has a k,, 2-80 s-I. The subsequent phase is accompanied by formation of the main product, 2-amino-5-oxocyclohex-1 -enecarboxyl CoA. This step consists in a hydrogenation of the primary, oxygenated and non-aromatic CoA intermediate; it has a rate ~1 . 3 s-', which is thus the second rate-limiting step in catalysis. As a side reaction of the oxidized enzyme and at low NADH concentrations the initially formed product disappears at a very slow rate (kbs = 0.05 s?). This third 'post-catalytic' process is not relevant for catalysis. The primary product 2-amino-5-oxocyclohex-I -enecarboxyl-CoA is dehydrogenated by the oxidized enzyme to yield the aromatic 2-amino-5-hydroxybenzoyl-CoA as secondary product. The reduced enzyme formed in this process is reoxidized by 0, to form H,O,. This explains the formation of different products depending on the actual concentration of NADH in the catalytic system, which has been reported previously [Buder, R., Ziegler, K., Fuchs, G., Langkau, B. & Ghisla, S. (1989) Eur J. Biochern. 185,. A kinetic mechanism is proposed based on the concept that aminobenzoyl-CoA monooxygenaselreductase has two active sites which catalyze independently monooxygenation and hydrogenation of substrate or intermediate.

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Section: Mechanistic Conclusionsupporting

confidence: 78%

Kinetic and Mechanistic Studies on the Reactions of 2‐Aminobenzoyl‐CoA Monooxygenase/Reductase

Langkau

Ghisla

1995

European Journal of Biochemistry

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“…The degradation of tannins in the absence of molecular oxygen has also been documented (Evans & Fuchs, 1988 ;Fuchs et al, 1994), and several ruminal microorganisms that can degrade phenolic monomers have been isolated. Eubacterium oxidoreducens degrades gallate, phloroglucinol and pyrogallol to acetate and butyrate in the presence of hydrogen and formic acid (Krumholz & Bryant, 1986).…”

Section: Introductionmentioning

confidence: 94%

Effect of hydrolysable and condensed tannins on growth, morphology and metabolism of Streptococcus gallolyticus (S. caprinus) and Streptococcus bovis

O’Donovan

Brooker

2001

Microbiology

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Streptococcus gallolyticus (S. caprinus) was resistant in vitro to at least 7 % (w/v) tannic acid and 4 % (w/v) acacia condensed tannin, levels 10-fold greater than those tolerated by S. bovis. Growth of S. gallolyticus in liquid medium was characterized by a lag period which increased, and a growth rate which decreased, with increasing tannin concentration. S. gallolyticus was also more tolerant to the presence of simple phenolic acid monomers than was S. bovis, but the lag period was still concentration dependent. Gallate decarboxylase activity in S. gallolyticus was elevated in the presence of tannic acid or gallic acid but not with other phenolic acids. Scanning electron microscopic analysis showed that both the size and shape of S. gallolyticus and S. bovis changed in response to tannin but only S. gallolyticus was surrounded by an extracellular polysaccharide matrix which accumulated in a tannin-concentration-dependent fashion. Washing of the cells to remove extracellular polysaccharide increased the lag period of S. gallolyticus in the presence of 1 % (w/v) tannic acid from 4 h to 6 h. In contrast, increasing extracellular polysaccharide synthesis in S. bovis did not increase its tolerance to tannic acid. These data demonstrate that S. gallolyticus has developed a number of mechanisms to reduce the potential effect of tannins on cell growth, and that these mechanisms provide the organism with a selective advantage over S. bovis when grown in the presence of tannins.

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“…However, fermenting bacteria convert benzoate according to reaction (3). The fermentation products are removed by methanogens living in syntrophy and tran formed to 3.75 moI CH, and 3.25 rnol CO,/mol benzoate accord g to reaction (4) Pyrophosphate is used for phoaphorylation.…”

Section: Disc Ljssi Onmentioning

confidence: 99%

Benzoyl‐Coenzyme A Reductase (Dearomatizing), a Key Enzyme of Anaerobic Aromatic Metabolism

Boll

Fuchs

1995

European Journal of Biochemistry

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212

255

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Anoxic metabolism of many aromatic compounds proceeds via the common intermediate benzoylCoA. Benzoyl-CoA is dearomatized by benzoyl-CoA reductase (dearomatizing) in a two-elcctron reduction step, possibly yielding cyclohex-1 ,S-diene-l-carboxyl-CoA. This process has to overcome a high activation energy and is considered a biological Birch reduction. The central, aromatic-ring-reducing enzyme was investigated for the first time in the denitrifying bacterium YhuuercL a~~rnaticu strain K172. A spectrophotometric assay was developed which was strictly dependent on MgATP. both with cell extract and with purified enzyme. The oxygen-sensitive new enzyme was purified 35-fold with 20% yicld under anaerobic conditions in the presence of 0.25 mM dithionite. It had ii native molecular mass of approximately 170 kDa and consisted of four subunits a,b,c,d of 48, 45, 38 and 32 kDa. The oligomer composition of the protein most likely is abcd. The ultraviolet/visible spectrum of the protein as isolated, but without dithionite, was characteristic for an iron-sulfur protein with an absorption maxiinuni at 279 nm and a broad shoulder at 390 nm. The estimated molar absorption coefficient at 390 nm was 35000 M-l cm-'. Reduction of the cnzyrne by dithionite resulted in a decrease of absorbance at 390 nm, and the colour turned from greenish-brown to red-brown. The cnzyrne contained 10.8 2 1.5 mol Fe and 10.5 1 1.5 mol acid-labile sulfur/mol. Besides zinc (0.5 mol/mol protein) no other metals nor selenium could be detected in significant amounts. The enzyme preparation contained a flavin or tlavin-like conipound; the estimated content was 0.3 mol/mol enzymc. The enzyme reaction required MgATP and a strong reductant such as Ti(II1). The reaction catalyzed i s : benzoyl-CoA f 2 Ti(lI1) f n ATP -nonaromatic acyl-CoA + 2 Ti(TV) + n ADP + n P,. The estimated number n of ATP molecules hydrolyzcdl two electrons transferred in benzoyKoA reduction is 2-4. In the absence of benzoyl-CoA the enzyme exhibited oxygen-sensitive ATPase activity. The enzyme was specific for Mg' ' -ATP, other nucleoside triphosphates being inactive (< 1 %). Mg" could be substituted to some cxtent by MnZ+, Fez+ and less efficiently by Co' ' . Benzoate was not reduced, whereas some fluoro, hydroxy, amino and methyl analogues of the activated benzoic acid were reduced, albeit at much lower rate; the products remain to be identified. The specific activity with reduced methyl viologen as the electron donor was 0.55 pmol min-' mg-' corresponding to a catalytic number of 1.6 s I. The apparent K , values under the assay conditions (0.5 mM for both reduced and oxidized methyl viologen) of benzoyl-CoA and ATP were IS pM and 0.6 mM, respectively. The enzyme was inactivated by ethylene, hipyridyl and, in higher concentrations, by acetylene. Benzoyl-CoA reductase also catalyzed the ATP-dependent two-electron reduction of hydroxylamine ( K , 0.15 mM) and azide. Some of the properties of the enzyme are reminiscent of those of nitrogenase which similarly overcomes the high activation en...

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Biochemistry of anaerobic biodegradation of aromatic compounds

Cited by 91 publications

References 209 publications

Kinetic and Mechanistic Studies on the Reactions of 2‐Aminobenzoyl‐CoA Monooxygenase/Reductase

Kinetic and Mechanistic Studies on the Reactions of 2‐Aminobenzoyl‐CoA Monooxygenase/Reductase

Effect of hydrolysable and condensed tannins on growth, morphology and metabolism of Streptococcus gallolyticus (S. caprinus) and Streptococcus bovis

Benzoyl‐Coenzyme A Reductase (Dearomatizing), a Key Enzyme of Anaerobic Aromatic Metabolism

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