Enoate reductases reactions

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. 

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. 

For example78. (2 )-2-methyI-3-phenylpropanate can be prepared in the described manner in 95 % yield using the enoate reductase from Clostridium LAI. Immobilization of the enzyme 7 gives no improvement in the reaction rates in fact, when free or immobilized cells are used the reaction becomes slower. A part of the current is used for hydrogen production. 

Table 2 reveals the multitude of catalysed reactions and Table 3 shows the surprising broad substrate specificity of enoate reductase from Clostridium tyrobutyricum DSM 1460. For a series of substrates Table 3 shows kinetic data (6,8,18,20,24). Only a few of these substrates can not be prepared in a sterical pure form with whole cells or crude extract from C. tyrobutyricum without additional measures. There are two aspects which have to be emphasized Whole cells contain a 2-enoyl-CoA reductase (EC 1.3.1.8) besides enoate reductase (EC 1.3.1.31) (Scheme 3). After offering ( )-2-butenoate, ( )-... 

Reactions catalysed by enoate reductase. The stereochemical course of reactions [9a]-[10b] is the same as that indicated for reaction [9],... 

SCHEME 3 A few 2-enoates (see text) are reduced by enoate reductase or cfter activation to a 2-enoyl-CoA-ester also by enoyl-CoA reductase. The stereochemical course of both reactions is different (25). In resting cells of C. tyrobutyricum the reaction of enoyl-CoA reductase can be blocked (Section 2.5.3). 

Substrates of enoate reductase that are reduced according to the reactions shown in Table 2. All CC-double bonds in addition to the a,P position are E configured. The relative rate 100 % corresponds to 22 U mg of purified enoate reductase at pH 6.0 and 0.25 mM NADH. The A m value for NADH is 0.012 mM and for reduced methylviologen 0.4 mM... 

Table 3 (No. 16) shows that ( )-p-nitro-cmnamate or derivatives can be reduced by enoate reductase, too. However, this reaction can be performed only with isolated enoate reductase using NADH as electron donor. Reduced methylviologen spontaneously reacts with nitro groups. Aliphatic as well as aromatic nitro compounds are also reduced by ferredoxins present in clostridia (26).

From a practical point of view very selective reactions are possible. As already mentioned the CC-double bond of /7-nitro-cinnamic acid can be reduced with enoate reductase and NADH without attacking the nitro group. 

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. 

Reaction [9] is inhibited by 0.4 mM morin or dicoiunarol to 80 and 60%, respectively. The same concentration of dicoiunarol inhibits Reaction [9a] by less than 5 % (17). These facts and other results (20) are indicative of three binding domains of enoate reductase one for NADH which can be blocked by dicoiunarol or morin, another for enoates which can be occupied by hunarate, and a third one for reduced methylviologen. For studies on the iron/sulphur centres see (21). 

From a mechanistic point of view, the behaviour of the P-halogenated enoates is of interest (24). (Z)-3-Chloro-cinnamate and (Z)-3-chloro-butenoate as well as (Z)-3-bromo-cinnamate consume in the presence of enoate reductase 2 mol NADH per mol instead of 1 mol as other enoates do. The products are the saturated halogen-free carboxylates (Reaction [14]). 

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). 

More than 10 experiments. Values of enantiomeric excess depend on temperature, reaction time etc.. The ee values decrease with increase of temperature and reaction time. A total volume of 50 ml contained 50 mM substrate, 1 mg ( 12 U) enoate reductase. After 17 h at 18 "C > 95 % product. A total volume of 4 ml contained 70 pmol ethanol, 1.4 pmol NADH, 100 mg C. klityveri or 1 U enoate reductase. After 22 h at 25 °C > 95 % product. The NADH contained about 5 % NAD, which is necessary for the dehydrogenation of the substrate to 2-methyl-3-phenyl-enal which is then reduced to the (R)-2-methyl-3-phenyl-propanol. Horse liver alcohol dehydrogenase, 2.5 U. After 22 h at 25 °C > 95 % product. 

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-... 

Reaction [13] in Table 2 is actually a consequence of the ability of enoate reductase to dehydrogenate saturated aldehydes with NAD. Kinetic and analytical studies showed that the apparent isomerisation is actually a redox reaction needing traces of NADH besides enoate reductase. The 2-methenyl-aldehyde is reduced by NADH and the NAD ... 

Dehydrogenations, racemizations, exchange of a-protons and rearrangements of aldehydes catalysed by enoate reductase. Depending on the rate of reaction different amounts of enzyme have been used. The racemizations have been studied at pH 7.0, the H/ H-exchange and the dehydrogenations at pH 8.0 (18). 

At pH 7.0 the aldehyde oxidoreductase does not any longer reduce carboxylates to aldehydes but the enoate reductase fi om C. tyrobutyricum is active (Section 2). Starting fi om fi-om a- and/or -substituted enoates the chiral saturated acids can afterwards be reduced fiirther with carbon monoxide and C. thermoaceticum to the corresponding chiral alcohols. An isolation of the chiral acid is not necessary. After reducing the 2-enoate (Reaction [9]) the pH has to be shifted to pH 5.0-5.5. Cells of C. thermoaceticum are added and the chiral carboxylate is reduced with carbon monoxide (74,77). The enoate reducing system of C. thermoaceticum shows a low activity and seems not to be as stereospecific as that of C. tyrobutyricum (unpublished). 

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.

A promising new area of enzymatic reactions is that of anaerobes. These organisms use redox systems that are not pyridine nucleotide dependent, but can use methylvirologen or benzylviologen (commercially available) as mediators. Electron donors can be hydrogen gas, formate, or carbon monoxide rather than glucose. The first new enzyme from this source is a 2-enoate reductase, effected with stereospecific /ro 5-addition of hydrogen. An... 

To circumvent these issues, instead of wild-type whole cells, the use of cloned and overexpressed enoate reductases together with suitable redox enzymes for cofactor recycling - the so-called designer bugs - appears as an excellent ahemative to overcome the lack of selectivity observed when whole (fermenting) cell systems are applied, due to the existence of enzymes that can catalyze side reactions, and so on (Scheme 2.4) [18,19]. Thus, when enoate reductases are overexpressed, the enhancement of its concentration leads to higher selectivity and yields in the desired products. This area is clearly expanding nowadays, and therefore further innovations are ejqjected to appear. 

The so-called redox isomerizations represent a dass of internal redox reactions tvherein the overall redox state of the starting material remains unchanged. Again, this type of reaction is ideally suited to couple two oxidoreductase-catalyzed reactions with opposite cofactor demand. We [83] and others [84] have demonstrated the redox isomerization of allylic alcohols into the corresponding saturated ketones by coupling an ADH with an enoate reductase (ER) (Scheme 8.18). 

Divergent reaction conditions are well exemplified in the case of reduction and oxidation steps. Consequently it is easier to run such systems sequentially [53]. This leads to the possibility of a linear-cyclic-parallel system for oxidation and reduction, proposed by Oberleitner and coworkers [54]. Here oxidative and reductive catalysts were combined to balance redox, followed by a further oxidation reaction. For example, alcohol dehydrogenase and enoate reductase, followed by Baeyer-Villiger monooxygenase, have been demonstrated in such a system. The last oxidation needs to be run with a parallel system or alternatively in a whole cell for cofactor recycle. 

NAD(P)H-dependent catalytic reduction reactions such as carbonyl or enoate reductases and hydroxy acid or amino acid dehydrogenases [14],... 

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