Reductions isolated enzymes

Recently, there have been new technologies described for multienzymatic processes and tools to evaluate them [71,72]. These approaches should also be incorporated in the evaluation of multienzymatic cascades, as used for cofactor regeneration in many isolated enzyme reductions. 

An isolated enzyme is also used for the reduction in SCCO2 [20cj. H LADH was used for reduction of butyraldehyde as shovm in Figure 8.27. In this case, addition of... 

Figure 8.27 Reduction of aldehyde in SCCO2 by an isolated enzyme, horse liver alcohol dehydrogenase (HLADH) [20c] (a) Reaction scheme (b) fluorinated coenzyme soluble in CO2 and (c) effect of coenzyme on the reaction.

The addition of sulfite to APS reductase results in changes of the flavin visible spectrum that are explained by the formation of an adduct between the sulfite and the FAD group (135). Addition of AMP to the as-isolated enzyme causes no change in the spectroscopic properties. Addition of AMP to the sulfite-reacted enzyme causes the reduction of center I. However, the formation of a semiquinone signal has never been observed either by EPR or visible spectroscopies. Also, Mossbauer and EPR data indicate that AMP closely interacts with center I (139). 

Another very recent development in the field of enzymatic domino reactions is a biocatalytic hydrogen-transfer reduction of halo ketones into enantiopure epoxides, which has been developed by Faber, Bornscheuer and Kroutil. Interestingly, the reaction was carried out with whole lyophilized microbial cells at pH ca. 13. Investigations using isolated enzymes were not successful, as they lost their activity under these conditions [26]. 

Goldberg, K., Schroer, K., Luetz, S. and Liese, A. (2007) Biocatalytic ketone reduction - a powerful tool for the production of chiral alcohols -part I processes with isolated enzymes. Applied Microbiology and Biotechnology, 76, 237-248. 

Hummel, W., Abokitse, K., Drauz, K. et al. (2003) Towards a large-scale asymmetric reduction process with isolated enzymes Expression of an (5)-alcohol dehydrogenase in E. coli and studies on the synthetic potential of this biocatalyst. Advanced Synthesis and Catalysis, 345 (1 + 2), 153-159. 

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. 

Most asymmetric reductions that can be enzymatically effected have been the reactions of ketones. These reactions can be conducted with whole cells as well as with isolated enzymes. In the latter case, of course, at least one equivalent of a cofactor such as NADH or NADPH (nicotinamide adenine dinucleotide) is required to serve as the actual reductant in the reaction system. 

The asymmetric reduction of prochiral functional groups is an extremely useful transformation in organic synthesis. There is an important difference between isolated enzyme-catalyzed reduction reactions and whole cell-catalyzed transformations in terms of the recycling of the essential nicotinamide adenine dinucleotide (phosphate) [NAD(P)H] cofactor. For isolated enzyme-catalyzed reductions, a cofactor recycling system must be introduced to allow the addition of only a catalytic amount (5% mol) of NAD(P)H. For whole cell-catalyzed reductions, cofactor recycling is automatically achieved by the cell, and the addition of a cofactor to the reaction system is normally not required. 

Analogous to the KRED reductions they can be performed as whole-cell biotransformations [48, 49] (baker s yeast, for example, contains a number of EREDs) or with isolated enzymes [50-52]. In the latter case the nicotinamide cofactor can... 

A systematic study on enzymatic catalysis has revealed that isolated enzymes, from baker s yeast or old yellow enzyme (OYE) termed nitroalkene reductase, can efficiently catalyze the NADPH-linked reduction of nitroalkenes. Eor the OYE-catalyzed reduction of nitrocyclohexene, a catalytic mechanism was proposed in which the nitrocyclohexene is activated by nitro-oxygen hydrogen bonds to the enzymes His-191 and Asn-194 [167, 168]. Inspired by this study Schreiner et al. 

Highly enantioselective reduction of ethyl 6-benzyloxy-3,5-dioxohexanoate by ADH of Acinetohacter calcoaceticus has been reported (97 to >99% ee) [6]. Regi-oselectivity was not encountered, however, as was the case in the reduction of a variety of 3,5-dioxohexanoates A with baker s yeast [7]. The application of isolated enzymes in an anticipated regio- and enantioselective reduction of diketo esters A seemed most promising to us. 

Reduction of a cyclobutanone with baker s yeast yielded the diastereomeric alcohols 5 which could be separated by column chromatography.163 The optical purity was reported to be ca. 90%. Stereoselective reductions using isolated enzymes or complete organisms have also been reported for 6.82-276 278... 

The tyrosyl radical is used to initiate the ribonucleotide reduction at the active site in the R1 protein 3.5 nm away. The tyrosyl radical is very stable and was discovered by a characteristic EPR spectrum of isolated enzyme. Alteration of this spectrum when bacteria were grown in deuterated tyrosine indicated that the radical is located on a tyrosyl side chain and that the spin density is delocalized over the tyrosyl ring.361 362 Using protein engineering techniques the ring was located as Tyr 122 of the E. coli enzyme. A few of the resonance structures that can be used to depict the radical are the following ... 

Many approaches have been used to deduce the sequence of carriers through which electron flow takes place (Fig. 18-5). In the first place, it seemed reasonable to suppose that the carriers should lie in order of increasing oxidation-reduction potential going from left to right of the figure. However, since the redox potentials existing in the mitochondria may be somewhat different from those in isolated enzyme preparations, this need not be strictly true. 

As mentioned before, the application of P450-catalyzed monoxygenations in organic synthesis is largely hampered by the insolubility/instability of most of the isolated enzymes of the family and by the complexity of the reduction system. In... 

As the use of dehydrogenases requires regeneration of the cofactor, employment of whole cells instead of isolated enzymes appears attractive growing or resting cells can recycle the cofactor internally so that no external addition is necessary. Table 19.4 lists some results of the use of whole cells in the reduction of keto compounds. 

Chiral alcohols are valuable products mainly as building blocks for pharmaceuticals or agro chemicals or as part of chiral catalysts. Cheap biotransformation methods for the selective reduction of particular ketone compounds are known for many years rather catalyzed by fermentation than with isolated enzymes. Products prepared with whole cells such as baker s yeast often lack high enantioselectivity and there were several attemps to use isolated enzymes. Resolution of racemates with hydrolases are known in some cases but very often the reduction of the prochiral ketone using alcohol dehydrogenases are much more attractive. 

In the use of whole cells severe problems may arise from strain specific activity of intracellular enzymes reacting with the product. For instance, the organism may use the substrate or the product as the carbon source or intracellular esterase activities may influence the yield of hydroxy esters formed by enzymatic reduction of keto esters. These problems may be avoided using isolated enzymes. These potential side reactions also define the purification grade of a technical enzyme sample because the complete separation of the disturbing activities must be ensured. 

Using isolated enzymes instead of whole cells, similar problems are to be considered only in a few cases. ADH from Thermoanaerobium brockii shows varying enantiomeric excess of the product depending on the structure of the ketone to be reduced. Conversions with this enzyme yield in products with low (20% for the reduction of acetophenone) or high ee value (100% for the reduction of p-Cl-acetophenone). Predictions about the stereospecificity of HLADH catalyzed reductions can be made for simple acyclic substrates applying Prelog s rule [37] and for more complex compounds using the cubic-space model developed by Jones and Jakovac [38],... 

As ascribed, the EPR spectrum with g = 2.10 can be low-spin Fec(III). When the isolated enzyme is reductively titrated this signal disappears at a potential Emj -0.3 V [65]. This would seem to indicate that the putative Fec(III) form is not relevant, at least not to hydrogen-production activity. The cubane is a one-electron acceptor as it can shuttle between the 2+ and 1 + oxidation states. Therefore, if the active center were to take up a total of two electrons, then the oxidation state of the Fec would, as least formally, shuttle between II and I. Recently, a redox transition in Fe hydrogenase with an Em below the H2/H+ potential has been observed in direct electrochemistry [89]. This superreduced state has not been studied by spectroscopy. It might well correspond to the formal Fec(I) state. For NiFe hydrogenases Fec(I) has recently been proposed as a key intermediate in the catalytic cycle [90] (cf. Chapter 9). 

Chapter 2 Reduction Reactions. In Preparative Biotransformations Whole Cell and Isolated Enzymes in Organic Synthesis, Roberts, S. M., Wiggins, K., Casy, G., Phythian, S. Eds., John Wiley Sons ... 

Enzymatic synthesis of E-tm-leucine is another example of the use of isolated enzymes (Bommarius et al, 1995). An NADH-dependent leucine dehydrogenase was used as a catalyst for the reductive amination of the corresponding keto acid together with formate dehydrogenase (FDH) and formate as a cofactor regenerator (Fig. 19.5b Shaked and Whitesides, 1980 Wichmann et al, 1981). Furthermore, a unique membrane reactor system involving FDH and PEG-modihed-NAD for continuous NADH regeneration... 

M(VI) and M(IV) oxidation states. The M(V) state is generated by a one-electron reduction of the M(VI) state, or the one-electron oxidation of the M(IV) state, and occurs during the catalytic cycle—en route to the regeneration of the catalytically active state. Spectroscopic studies of the Mo—MPT enzymes, notably electron spin resonance (EPR) investigations of the Mo(V) state, have clearly demonstrated that the substrate interacts directly with the metal center (37). The first structural characterization of a substrate-bound complex was achieved for the DMSOR from Rhodobacter capsulatus DMS was added to the as-isolated enzyme to generate a complex with DMSO that was O-bound to the molybdenum (43). 

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