Enoate reductases reductions

Enoate Reductase in Reduction of Triply Substituted Double Bonds... 

Enoate reductase reduces a,/3-unsaturated carboxylate ions in an NADPH-dependent reaction to saturated carboxylated anions. Useful chiral synthons can be conveniently prepared by the asymmetric reduction of a triply substituted C—C bond by the action of enoate reductase, when the double bond is activated with strongly polarizing groups [22]. Enoate reductases are not commercially available as isolated enzymes therefore, microorganisms such as baker s yeast or Clostridium sp. containing enoate reductase are used to carry out the reduction reaction. 

Another approach to preparing enantiomerically pure carboxylic acids and related compounds is via enanhoselective reduction of conjugated double bonds using NAD(P)H-dependent enoate reductases (EREDs EC 1.3.1.X), members of the so-called Old Yellow Enzyme family [44]. EREDs are ubiquitous in nature and their catalytic mechanism is well documented [45]. They contain a catalytic flavin cofactor and a stoichiometric nicotinamide cofactor which must be regenerated (Scheme 6.23). 

Simon and collaborators have described a novel stereoselective electroenzymatic reduction of alkenes of type 23 (equation 12)28. The enzyme enoate reductase is the reductant... 

Enoate reductase [153, 154], which occurs in strains of Clostridium or Proteus, and 2-oxo-acid reductase [155] from Proteus vulgaris or P. mirabilis catalyzes the stereospecific reduction of substrates performed directly by reduced methyl-viologen. No nicotinamide coenzyme is required. Methylviologen is regenerable electrochemically. Examples of the reduction of enoates, ketones, and 2-oxo acids are given in [155]. 

The generation of stereogenic centers by asymmetric reduction of carbon-carbon double-bonds is a current topic in chemoenzymatic synthesis. Though enzymes of the old yellow enzyme (OYE) family were identified to perform alkene reduction and were characterized some years ago [133-135], applications of enoate reductases in natural product syntheses are still rare. Thus, potential applications are also shown in this chapter. With an increasing number of new enoate reductases, such as YqjM reductase from B. subtilis, more and more possible targets for biotransformations can be found. 

Reductions catalyzed by enoate reductase enantioselective hydrogenation of a, /6-unsaturated enoates and comparable compounds. 

The enzyme enoate reductase has been isolated and described76,11. It is a conjugated iron sulphur flavoprotein. For preparative purposes purified enoate reductase can be applied in electrochemical cells7S. In this system methyl viologen (or another artificial electron mediator for the electron transfer) is continuously regenerated by an electrode and serves as an electron donor for the reductive reaction as shown in the following diagram. 

Aliphatic acylates show no measurable inhibition, if they are present at concentrations of 100-fold the values of the corresponding enoates. Measurable inhibitions can be observed with phenyl group-containing acylates. 3-Phenylpropionate in concentrations of 38 mM shows about 86 % inhibition. Fumarate, which is only a poor substrate, inhibits the reduction of enoates by NADH as well as by reduced methylviologen (Reactions [9] and [9a]). However, the reduction of NAD by reduced methylviologen (Reaction [11]) is not inhibited by fumarate. Interestingly, inhibitors such as morin or dicoumarol, which probably bind to the flavin domain of enoate reductase, do not impair the reduction of enoates by reduced methylviologen, but all reductions with NADH are inhibited. 

Enoate reductase exclusively splits off the (4S)-hydrogen atom from NADH. There is no direct hydrogen transfer from NADH to the products. If (45)-[ H]-labelled NADH with a total tritium activity of 4.67.10 decays per minute was dehydrogenated with an excess of enoate, the isolated product showed a tritium content of less than 0.005 % of that of the NADH. Almost all the tritium was in the water. In the absence of an enoate as acceptor, the tritium exchange from (45)-[4- H]-NADH catalysed by enoate reductase is very slow. Depending on the substrate concentration, the isotope effect of the reduction of ( )-2-methyl-2-butenoate with (4S)-[4- H]-NADH varies from 6.8 to 1.3. The presence of NAD" decreases the isotope effect (17). 

As shown in Table 6 enoate reductase or clostridia containing this enzyme (C. tyrobu-tyricum or C. kluyveri) catalyse the reduction of a,P-unsaturated aldehydes (enals) (Table 2 Reactions [10a] and [10b]). In contrast to saturated carboxylates, saturated aldehydes can be dehydrogenated to a,p-unsaturated aldehydes (enals) by enoate reductase in the presence of electron acceptors such as oxygen or dichlorophenol-indophenol, (Table 7, and Table 2 Reactions [12a] and [12b]), (18). 

Reductions of unsaturated aldehydes and unsaturated alcohols with enoate reductase. All of the here mentioned products possess (R)-configuration (18). 

The first step is catalysed by the pyridine nucleotide dependent alcohol dehydrogenase (NAD -dependent in C. kluyveri and NADP -dependent in C. tyrobutyricum) leading to the 2-enal which in turn is reduced by enoate reductase to the saturated aldehyde (Reactions [10a] and [10b]). The saturated aldehyde is further reduced to the alcohol. The rate of the reduction depends not only on the activity of the involved en2ymes but also on the concentration and on the ratio of NAD(P) /NAD(P)H. In the presence of MV which is formed by the reduction of MV by the system H2/hydrogenase (Reaction [5a]), the ratio NAD(P) /NAD(P)H is too small for the fast and complete dehydrogenation of an allyl alcohol since the first step of the reaction sequence [16], which needs NAD(P), is too slow. It turned out that ethanol is a better electron donor than hydrogen gas in this case. For the reduction of 50-70 mM ( )-2-methyl-2-butenol to (R) -2-methyl-... 

This treatment is especially recommended if the cells are lyophilised for deuterations. The cells can be lyophilised without loss of hydrogenase and enoate reductase activity. Without this treatment usually 60-80 % of the enzyme activity can get lost during freeze drying, even if contact with oxygen is tried to omit. Freeze dried cells are used for reductions in H20. 

Depending on the substrate the productivity number for the reduction of many enoates with cells of C. tyrobutyricum is in the range of 400-1500. The optimal temperature is 35-37 C, the optimal pH is 6.0-6.2. Purified enoate reductase shows a pH maximum at... 

Reductions of enoates with enoate reductase or with resting cells in an electrochemical cell allows to study the influence of various parameters from one single experiment (Section 6). 

Enoate reductase, 2-hydroxy carboxylate viologen oxidoreductase (HVOR) and AMAPORs (Section 5) are enzymes able to accept reversibly single electrons fi-om artificial mediators such as viologens and others. These mediators transfer electrons fi om or to electrodes. Therefore by the presence of the aforementioned enzyme activities biocata-lytic redox reactions can be carried out in electrochemical cells. As already mentioned in Section 1.2 AMAPORs catalysing Reactions [8] and/or [8a] are rather ubiquitous. Electromicrobial reductions can also be carried out with yeasts (Section 1.2). Since the potential of the working electrode can be chosen at will, reductions as well as... 

Fig. 5. Electroenzymic reduction of (E)-2-methylcinnamate. The cathode compartment of an electrochemical cell contained 85 ml 0.1 M potassium phosphate buffer pH 7, 6.0 mmol 2-methylcinnamate and 0.28 mmol methylviologen. The working potential was set to -760 mM versus SCE and after the reduction of the methylviologen the reaction was started by adding 15 U enoate reductase. A maximum current of 40 mA was recorded. The drop of the current to almost zero was indicative for the complete conversion of the substrate to R)-2-methyl-3-phenylpropanoate.

Enoate reductase. Enoate reductase isolated from Clostridium tyrobutyricum catalyses the NADH or methylviologen-dependent reduction of the a,p carbon-carbon double bond of non-activated 2-enoates and in a reversible way that of 2-enals. The enzyme appears to be a multimer of identical subunits. The total molecular mass is 940 kDa ( 73 kDa per subunit). Sedimentation equilibrium experiments, molecular mass data, and electron microscopy indicate that the native enzyme is composed of a tetramer of trimers. Each enzyme subunit contains one FAD, 0.6 FMN and one [4Fe-4S] cluster. 

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