Enoate Reductase Research Articles

Photochemical, Electrochemical, and Hydrogen‐Driven Enzymatic Reductions in Reversed Micelles

AbstractA few examples of (multi)enzymatic reactions in reversed micellar media are described and discussed. The key enzymes in these examples are 20β‐hydroxy‐steroid dehydrogenase, enoate reductase, and an NAD+‐independent hydrogenase, catalyzing the reactions of interest, viz., the site‐ and stereo‐specific reduction of 20‐ketosteroids to their corresponding 20β‐hydroxy form, the chiral hydrogenation of α,β‐unsaturated carboxylates, and the production of hydrogen, respectively. These cofactor‐requiring enzymes are coupled to various in situ cofactor‐regenerating systems, powered either by light, electricity, or hydrogen. The performance of all these systems is shown.

Structure of Enoate Reductase from aClostridium tyrobutyricum (C. spec.La1)

Enoate reductase from Clostridium tyrobutyricum was purified by a rapid novel procedure. Chromatography on DEAE-Sepharose and on hydroxyapatite resulted in a high yield of about 90% pure enzyme in less than 10 h. A purity greater than 98% could be obtained by additional chromatography on Sephacryl S-300. The enzyme sediments in the analytical ultracentrifuge as a single, symmetrical boundary with a velocity of S(0)20,w = 24.9 S. Equilibrium ultracentrifugation yielded a molecular mass of 940 000 +/- 20 000 Da. The enzyme contains one type of subunit as shown by dodecyl sulfate electrophoresis and partial sequence determination. A subunit molecular mass of about 73 000 Da was established by dodecyl sulfate electrophoresis and by sedimentation equilibrium analysis in guanidine hydrochloride. In addition to FAD, iron and labile sulfur, the enzyme purified by the new method showed approximately 0.7 mol of FMN per mol of subunit. A dissociation product sedimenting at a velocity of S(0)20,w = 9.8 S can be obtained by various experimental protocols. The fragment was obtained in pure form by gel permeation chromatography. The molecular mass was 230 000 +/- 10 000 Da as shown by sedimentation equilibrium analysis. Thus it appears that the dissociation product is a trimer of the 73 000-Da subunit. The formation of the 10-S fragment by dissociation of the native enzyme is accompanied by the loss of most of the FMN, whereas the FAD content is not changed. The fragment catalysed the reduction of acetylpyridine adenine dinucleotide by NADH. However, enoate reductase activity with NADH or methylviologen as cosubstrate was low. Electron micrographs of negatively stained enoate reductase show trigonal symmetry. The data suggest that enoate reductase is a dodecamer (tetramer of trimers) with tetrahedral symmetry.

Chiral products from non-pyridine nucleotide-dependent reductases and methods for NAD(P)H regeneration.

Enoate reductase (EC 1.3.1.31) from a Clostridium tyrobutyricum strain catalyses the stereospecific reduction of many different alpha, beta-unsaturated carboxylates, aldehydes and even some ketones. The enzyme accepts electrons from NADH and, 1.5 times faster, from reduced methyl viologen (1,1'-dimethyl-4,4'-bipyridinium). Another new type of non-pyridine nucleotide-dependent reductase has an extremely broad substrate specificity for 2-oxo-carboxylates and 2-oxo-dicarboxylates. In crude extracts from Proteus mirabilis and Proteus vulgaris, specific activities of 2-12 mumol product formed per mg protein per min can be found when reduced methyl or benzyl viologen is used as electron donor. The products are (2R)-hydroxy acids. Enoate reductase and 2-oxo-carboxylate reductase are suitable for electro-enzymic reductions in which catalytic amounts of viologens are continuously reduced in an electrochemical cell. This procedure has three advantages: (1) regeneration of NAD(P)H by a second enzyme and substrate is not required, (2) the unstable pyridine nucleotides are not required in the reaction mixture, and (3) the rate of the reaction can be observed continuously by measuring an electric current. Several yeasts, as well as aerobic and anaerobic bacteria, catalyse the reduction of NAD(P)+ by reduced methyl viologen. Such cells can be used for electro-microbial reductions when only pyridine nucleotide-dependent reductases are present. Information about the enzymes which catalyse the reduction of NAD(P)+ at the expense of reduced methyl viologen is given.

Immunological relationship of enoate reductases from different clostridia and the classification ofClostridiumspecies La 1

Immunological relationship of enoate reductases from different clostridia and the classification of<i>Clostridium</i>species La 1

Immunological relationship of enoate reductases from different clostridia and the classification of Clostridium species La 1

Immunological relationship of enoate reductases from different clostridia and the classification of Clostridium species La 1

On the occurrence of enoate reductase and 2-oxo-carboxylate reductase in clostridia and some observations on the amino acid fermentation by Peptostreptococcus anaerobius.

Enoate reductase present in Clostridium kluyveri and Clostridium spec. La 1 could be detected in three strains of C. tyrobutyricum and ten clostridia belonging to the groups of proteolytic and saccharolytic or proteolytic species, respectively. In C. pasteurianum, C. butyricum and C. propionicum enoate reductase could not be found even after growth on (E)-2-butenoate. A 2-oxo-carboxylate reductase was present in rather low activities in the non-proteolytic clostridia which produce enoate reductase. High activities (up to 10 U/mg protein) of 2-oxo-carboxylate reductase were found in six of ten proteolytic clostridia. The substrate specificities of the enoate reductase and the 2-oxo-carboxylate reductases from the proteolytic clostridia were determined with different alpha, beta-unsaturated carboxylates (enoates) and 2-oxo-carboxylates, respectively. Enoates as well as 2-oxo-carboxylates are intermediates of the pathway by which amino acids are degraded. An explanation is offered for the long known but not understood fact that in the Stickland reaction isoleucine always acts as an electron donor and leucine and phenylalanine can be electron acceptors as well as donors. Peptostreptococcus anaerobius converting some amino acids to the same products as C. sporogenes did this also with the intermediates which were found for the reductive deamination of amino acids in C. sporogenes, however, in crude extracts reduction of enoates occurred only in an activated form.

On the kinetics and mechanism of enoate reductase.

Enoate reductases from Clostridium spec. La 1 and Clostridium kluyveri show a rather broad substrate specificity i.e. many alpha,beta-unsaturated carboxylates are reduced in a NADH-dependent reaction. The relative rates for different substrates are different for both reductases. The Km value of NADH for the reductase from C. spec. La 1 is about 12 muM. The transhydrogenase activity (reduction of N-acetylpyridine adenine dinucleotide) with NADH shows a maximum at pH 8 which is about 2 units higher than that for the reduction of enoates. Results of initial rate studies can be best explained by a Bi Bi ping pong mechanism. No back reaction and no proton exchange from 2(-3)H-labelled acylates could be demonstrated. NAD+ is a mixed-type inhibitor. The product inhibition constant Ki = 0.84mM and the dissociation constant for the dead-end inhibition complex 4.8mM. Aliphatic acylates show no measurable inhibition when they are applied in concentrations at the 100-fold Km values of the corresponding enoates. Measurable inhibitions can be observed with phenyl group-containing acylates. 3-Phenylpropionate (38mM) shows about 86% inhibition. Fumarate which is not a substrate inhibits the reduction of enoates by NADH as well as by reduced methylviologen. However, the reduction of NAD+ by reduced methylviologen as well that of acetylpyridine adenine dinucleotide by NADH is not inhibited by fumarate. On the other hand inhibitors such as morin or dicoumarol which probably bind to the flavin domain do not impair the reduction of enoates by reduced methylviologen however, all reductions with NADH are inhibited. These results are indicative for three binding domains: one for NADH which can be blocked by dicoumarol or morin, another for enoates which can be occupied by fumarate and a third one for reduced methylviologen. Enoate reductase splits off exclusively the (4S)-hydrogen atom from NADH. There is no direct hydrogen transfer from NADH to the products. Depending on the substrate concentration the isotope effect of the reduction of (E)-2-methyl-2-butenoate with (4S)-[4(-3)H]NADH varies from 6.8 to 1.3. The presence of NAD+ decreases the isotope effect.

Electro‐Enzymatic and Electro‐Microbial Stereospecific Reductions

The hydrogenation of prochiral α,β‐unsaturated carboxylates and aldehydes to chiral compounds can be accomplished electrochemically, both with isolated enoate reductase as well as with whole bacteria cells which contain this enzyme; methylviologen functions as electron‐transfer agent. Since NAD+ can also be reduced to NADH, in principle all substrates of NAD‐dependent enzymes can be stereospecifically hydrogenated with this method.

Steric course of the NIH shift in the enzymic formation of homogentisic acid.

1 4-Hydroxyphenylpyruvate dioxygenase has been purified from beef liver to a specific activity of 0.021 U/mg at 25°C. 2 Samples of (3R)- and (3S)-4′-hydroxyphenyl[3-2H1]pyruvate were prepared by taking advantage of the known stereospecificity of phenylpyruvate keto—enol-isomerase (tautomerase). These were converted in situ by the dioxygenase into the corresponding deuterium-labelled homogentisic acids, which were isolated as the crystalline 3′,5′-dimethyl derivatives after treatment with dimethyl sulphate. 3 2′,5′-Dimethoxy-(E)-cinnamate was deuterated by enoate reductase from Clostridium sp. La 1 to afford (2S, 3R)-3-(2′,5′-dimethoxyphenyl)-[2-2H1, 3-2H1]propionate which was in turn chemically degraded to (S)-2,5-dimethoxyphenyl[2H1]acetic acid. 4 After the chiroptical characterisation of the chiral 2,5-dimethoxyphenyl[2H1]acetic acid had failed, the following samples of 2,5-dimethoxyphenylacetic acids were converted into the (–)-menthyl ester: (a) unlabelled, (b) statistically deuterated in position 2 (and in the aromatic positions), (c) (2S)-[2-2H1] as reference, (d) produced by the dioxygenase from (3R)-4′-hydroxyphenyl[3-2H1]pyruvate, and (e) produced by the dioxygenase from (3S)-4′-hydroxyphenyl[3-2H1]pyruvate. 5 In the 500-MHz 1H-NMR spectrum the 2-methylene protons of unlabelled 2,5-dimethoxyphenylacetic acid (–)-menthyl ester appeared as a well resolved AB system. Using a triple resonance technique and the reference samples (b) and (c) the 2-HRe atom could be correlated with the high-field half and the 2-HSi atom with the lowfield half of the AB system. 6 By comparison of the spectra of the enzymically obtained samples (d) and (e) with the reference spectra it was concluded that in the dioxygenase reaction the side-chain migration occurred with retention of configuration at the methylene C atom.

The reduction of allyl alcohols by Clostridium species is catalyzed by the combined action of alcohol dehydrogenase and enoate reductase.

Cells, as well as crude extracts of Clostridium kluyveri or Clostridium spec. La 1, catalyze the hydrogenation of (E)- or (Z)-2-butenol to n-butanol. No single enzyme could be detected which directly accomplishes this reaction. It turned out that the reduction occurs as follows: 2-butenol leads to 2-butenal leads to n-butanal leads to n-butanol. The first step is catalyzed by the NAD-dependent alcohol dehydrogenase in C. kluyveri, the second by the recently detected enoate reductase which reduces not only nonactivated alpha, beta-unsaturated acylates but also alpha, beta-unsaturated aldehydes in a NADH-dependent reaction and the third step is again catalyzed by alcohol dehydrogenase. In Clostridium La 1 the alcohol dehydrogenase is NADP-dependent. The rate of the reduction of 2-butenol to n-butanol depends not only on the enzymes, but also on the ratio NAD(P)/NAD(P)H. In the presence of methylviologen cation radical which is formed by the reduction of methylviologen by the system H2/hydrogenase, the ratio NAD(P)/NAD(P)H is too small for the dehydrogenation of 2-butenol to 2-butenal. This explains the antagonistic effect of methylviologen in the hydrogenation of allyl alcohols and 2-enoates by both Clostridium species. Furthermore, the mechanism explains the finding that from a preparative point of view ethanol is a better electron donor than hydrogen for the stereospecific reduction of allyl alcohols.

The activities of hydrogenase and enoate reductase in two Clostridium species, their interrelationship and dependence on growth conditions.

The enzyme activities of Clostridium La 1 and Clostridium kluyveri involved in the stereospecific hydrogenation of α,β-unsaturated carbonyl compounds with hydrogen gas were measured. In C. La 1 the specific activities of hydrogenase and enoate reductase depended heavily on the growth phase and the composition of the medium. During growth in batch cultures on 70 mM crotonate the specific activity of hydrogenase increased and then dropped to about 10% of its maximum value, whereas the activity of enoate reductase reached its maximum in cells of the stationary phase. Under certain conditions during growth the activity ratio hydrogenase: enoate reductase changed from 120 to 1. Thus, the rate limiting enzyme for the hydrogenation can be either the hydrogenase or the enoate reductase, depending on the growth conditions of the cells.

Stereospecific hydrogenations with immobilized microbial cells or enzymes

Stereospecific hydrogenations according to the general scheme [formula: see text] are of interest from a preparative and mechanistic point of view. Proteus mirabilis is suitable for the hydrogenation of a-keto-acids to R-hydroxy-acids, and a Clostridium strain for the reduction of enoates. Both have been immobilized in formaldehyde crosslinked gelatin and the latter also in polyacrylamide. Immobilized as well as free cells showed usually half lives of 100-200 h. The immobilized cells could be separated from the products and reused. In order to hydrogenate enoates stereospecifically, formate dehydrogenase and enoate reductase have been separately immobilized and coimmobilized on controlled pore glass. The yields for the separately immobilized enzymes were about 30 per cent and 70-80 per cent, respectively. The measured rate of the coupled system with immobilized enzymes was compared with the calculated rate, taking into account effects of pore diffusion for the pyridine nucleotide. Under operational conditions the half-life of the immobilized formate dehydrogenase was 36 h versus 45 h for the free enzyme. The corresponding values for the enoate reductase turned out to be about 17 h versus about 15 h. So far the immobilized as well as the free enzymes seem to be less stable than immobilized or free cells.

Occurrence and the possible physiological role of 2-enoate reductases

Recently we described the purification and some properties of a hitherto unknown NADH-dependent enzyme from the Clostridium species La 1 growing on (E)2-butenoate which reduces the carbon-carbon double bond of a&unsaturated carboxylate anions (2-enoates) [ 11. We now report a purified enoate reductase from Clostridium kluyveri and the occurrence of such an activity in the typical amino acid fermenting species Clostridium sporogenes. So far no physiological role of the reductase is known. In the metabolic schemes of clostridia fermenting (E)2-butenoate or ethanol and acetate such an enzyme was not postulated [2]. Up to 1.5% of the soluble protein of the Clostridium sp. La 1 grown under certain conditions consists of the reductase [3,41. In C. sporogenes we found enzyme activities necessary for the following reaction sequence: