Enzymatic formation acetaldehyde

The biogenesis of solerone 1 and related compounds was successfully rationalized by biomimetic model reactions. As key step we established the pyruvate decarboxylase catalyzed acyloin condensation of pyruvic acid with ethyl 4-oxobutanoate 4 or ethyl 2-oxoglutarate 3 with acetaldehyde. The importance of the ethyl ester function in 3 and 4 serving as substrates for the enzymatic formation of a-hydroxy ketones 5 and 6 was demonstrated. The identification of six yet unknown sherry compounds including acyloins 5 and 6, which have been synthesized for the first time, confirmed the relevance of the biosynthetic pathway. Application of MDGC-MS allowed the enantiodifferentiation of a-ketols and related lactones in complex sherry samples and disclosed details of their biogenetic relationship. 

Growth conditions in deep-well microtiter plates were optimized with respect to optimal expression of active enzymes (Fig. 2.2.1.1). The best results were obtained with an expression time of 20 h at 37 °C (Fig. 2.2.1.1, lanes 7-9). Subsequently, E. coli cells were enzymatically disrupted by lysozyme treatment, and the carboligase activity was monitored by a modified tetrazolium salt color assay [16], This color assay is based on the reduction of the 2,3,5-triphenyltetrazolium chloride (TTC) 13 to the corresponding formazan 15, which has an intense red color (Fig. 2.2.1.2A). Before screening ofa BFD variant library, substrates and products were tested in the color assay. Neither substrate, benzaldehyde 4 nor dimethoxy-acetaldehyde 8, reduced TTC 13 however, the product 2-hydroxy-3,3-dimethoxy-propiophenone 10 already caused color formation at low concentrations of 2.5-10 mM (Fig. 2.2.1.2B). Benzoin 12 as the product also gave a color change at a similar concentration (data not shown). 

First steps to elucidate the reaction mechanism of PDC were achieved by the investigation of model reactions using ThDP or thiamine [36,37], Besides the identification of C2-ThDP as the catalytic center of the cofactor [36], the mechanism of the ThDP-catalyzed decarboxylation of a-keto acids as well as the formation of acyloins was explained by the formation of a common reaction intermediate, active acetaldehyde . This active species was first identified as HEThDP 7 (Scheme 3) [38,39]. Later studies revealed the a-carbanion/enamine 6 as the most likely candidate for the active acetaldehyde [40 47] (for a comprehensive review see [48]). The relevance of different functional groups in the ThDP-molecule for the enzymatic catalysis was elucidated by site-directed substitutions of the cofactor ThDP by chemical means (for a review see... 

Formation of hybrid adducts with acetaldehyde and malondialdehyde (MAA adducts) has been shown to act in a synergistic manner and may be involved in the stabilisation of protein adducts in vivo. Malondialdehyde (HOCH=CH-CHO) is a highly reactive dialdehyde originating from the non-enzymatic lipid peroxidation of a variety of unsaturated fatty acids. 

The biosynthesis of optically active TIQs, exemplified by the presence of ( )-salsolinol in human fluids (189b), requires some comment. This is particularly warranted since many investigators have concluded that the conversion of dopamine to TIQ 64b is the result of condensation of the amine with acetaldehyde originating directly from ethanol. As shown in Fig. 32, this reaction, when carried out in vitro, affords racemic salsolinol (64) (5a,10). Formation of optically active TIQ 64b from dopamine and acetaldehyde would require that the Pictet-Spengler reaction be enzymatically controlled, as observed in the condensation of dopamine with 4-hydroxyphenylacetaldehyde in benzylisoquinoline-producing plants (209). The enzyme required to perform this reaction in mammalian systems has not yet been found. There are several observations which dispute such a reaction taking place in mammals the finding of 1-carboxy-TIQ 91 and DIQ 69 as major metabolites (189) and the very low levels of acetaldehyde detected in the brains of animals after alcohol consumption (215,216). This makes the acetaldehyde route to optically active 1-methyl-substituted TIQ suspect. 

The reaction of optically active carbinolamines formed by an enzymatically controlled addition of acetaldehyde to amines, illustrated in Fig. 2, may be of theoretical interest, but lacks experimental verification it also would require the presence of acetaldehyde. The more likely pyruvic acid route to optically active TIQs, however, also remains inconclusive. If it indeed proceeds through TIQ-1-carboxylic acids to DIQ intermediates by an oxidative decarboxylation (176,217,218), it requires that it be followed by an asymmetric enzymatic reduction. Although achieved in vitro (35), this reaction has not been realized in vivo. The formation of unequal amounts of the optical isomers of salsolinol and other TIQs in vivo could arise from racemic 1-carboxy-TIQ in an enzymatic decarboxylation, proceeding with (S) and (R) enantiomers at a different rate and thus affording different amounts of (5)- and (/ )-TIQ. With the availability of optically active TIQ-1-carboxylic acids, this possibility can now be tested. 

Ru(bpy)3" ]. This reduced species is oxidized by Rh(bpysa).r, resulting in the formation of the reduced hydridorhodium complex, which provides further reduction of NAD" ". The NADH regenerated in this photochemical system was coupled with the enzymatic reduction of acetaldehyde to ethanol in the presence of alcohol dehydrogenase. 

Enzyme Cofactors- In many enzymatic reactions, and in particular biological reactions, a second substrate (i.e., species) must be introduced to activate the enzyme. This substrate, which is referred to as a cofactor or coenzyme even though it is not an enzyme as such, attaches to the enzyme and is most often either reduced or oxidized during the course of die reaction. The enzyme-cofactor complex is referred to as a holoenzyme. The inactive form of the enzyme-cofactor complex for a specific reaction and reaction direction is called an apoenzyme. An example of the type of system in which a cofactor is used is the formation of ethanol from acetaldehyde in the presence of the enzyme alcohol dehydrogenase (ADH) and the cofactor nicotinamide adenine dinuoleotide (NAD) ... 

To measure ethanol in blood, enzymatic analysis is the method of choice for many laboratories. In this method, ethanol is measured by oxidation to acetaldehyde with NAD, a reaction catalyzed by alcohol dehydrogenase (ADH). With this reaction, formation of NADH, measured at 340nm, is proportional to the amount of ethanol in the specimen. The reaction is driven almost completely to the right by use of excess NAD and ADH and agents such as semicarbazide or tris (hydroxymethyl) aminomethane to trap acetaldehyde as it is formed. ... 

The hydroxylation of tryptophan produces 5-hydroxytryptophan, which can then be decarboxylated, catalyzed by tryptophan decarboxylase, a PALP-requiring enzyme, to 5-hydroxy tryptamine, also known as serotonin. Serotonin is an important compound in normal brain function and tranquility. Therefore, any disturbance of tryptophan metabolism via this pathway can lead to mental disturbances. Serotonin can be destroyed by the enzyme monoamine oxidase (a flavo protein), which catalyzes the formation of ammonia and 5-hydroxyindole acetaldehyde in an irreversible reaction. The aldehyde is rapidly oxidized enzymatically, utilizing NAD+ to form 5-hydroxy indoleacetate, which is then usually excreted. The formation and turnover of serotonin can be estimated by 5-hydroxy indoleacetate output in the urine. 

In the enzymatic aldol reaction, the role of the donor and acceptor is strictly determined by the specificity of the enzyme and only raie coupling product can be obtained. In contrast, the possible product range is more complex in acyloin and benzoin reactions If only a single aldehyde species is used as substrate, only one product can be obtained via homocoupling however, a pair of regioisomeric a-hydroxyketones can be obtained via heterocoupling, when two different aldehydes are used, the ratio of which is determined by the choice of substrates (e.g., benzoyl formate vs. benzaldehyde, pyruvate vs. acetaldehyde), and the specificites of enzymes, respectively (Scheme 2.198). 

Model reactions of this type have been studied in which the catalyst is pyridoxal plus a metal. The enzymatic reactions all appear to use pyridoxal phosphate as a cofactor, and in the case of a bacterial system, Mn" is also required. A major difference between the enzymatic and the model reactions is the requirement for a folic acid cofactor in the former. The formation of glycine and acetaldehyde from L-threonine and L-allo-threonine has been described by Lin and Greenberg. Their partially purified enzyme, threonine aldolase, was not shown to require any cofactors, and the reaction was not reversed. This is in contrast to the results of nonenzymatic experiments in which pyridoxal and a metal catalyze the reversible cleavage of threonine. 

Three mechanisms, at least, exist for the formation of AMC enzymatically. These are (1) from 2 moles of acetaldehyde, (2) from 1 mole of pyruvate and 1 mole of acetaldehyde, and (3) from 2 moles of pyruvate. 

---