Carboligase reaction

In order to increase the understanding of ThDP-dependent enzymes, the identification of amino acid side chains important for the catalysis of the carboligase reaction in pyruvate decarboxylase from Zymomonas mohilis (E.C. 4.1.1.1) and benzoylformate decarboxylase from Pseudomonasputida (E.C. 4.1.1.7) was a major task. Using site-directed mutagenesis and directed evolution, various enzyme variants were obtained, differing in substrate specificity and enantioselectivity. 

As demonstrated in Scheme 3, the ThDP-bound active aldehyde 6 as an acyl-donor may be added to a second aldehyde cosubstrate (acyl-acceptor) in an acyloin condensation-type reaction. This carboligase reaction was intensively investigated with acetaldehyde as an acyl-donor, which may be either condensed to a further acetaldehyde molecule yielding acetoin [1,26,27,29,63,153,154] or to a wide range of various aliphatic, aromatic and heterocyclic aldehydes [5,14,118,151,154-157,161]. 

A detailed investigation of the carboligase reaction mediated by PDC from yeast, wheat germ and Z. mobilis revealed that the stereo-control of this reaction is only strict with aromatic aldehydes as acylanion-acceptors, while the formation of acetoin (3-hydroxybutan-2-one) resulted in mixtures of the (R)- and (S)-enantiomer. 

The improvement of the carboligase reaction of PDCZ.m. is a further example for the potency of the rational approach in designing enzymes with... 

Further experiments were undertaken to investigate the possible participation of thiamine pyrophosphate in the carboligase reaction.28 Addition of TPP, Mg24, and pyruvate to the incubation mixture gave no... 

The optimal feeding profile based on the model is shown in Figure 3 and the simulation profiles are shown in Figure 4 for initial substrate concentrations of 90 mM benzaldehyde and 108 mM sodium pyruvate, and initial PDC activity of 4.0 U ml carboligase. Feeding was programmed at hourly intervals and the initial reaction volume would increase by 50% by the end of the simulated biotransformation. 

Simulation profile of fed batch PAC biotransformation kinetics at 6°C with initial PDC activity of 4.0 U carboligase ml, 90 mM benzaldehyde and 108 mM sodium pyruvate. Feeding was performed hourly as illustrated in Fig. 3 and the initial reaction volume of 30 ml (which would be used experimentally) increased to 45 ml at the end of reaction. 

Fed- batch PAC biotransformation kinetics at 6°C with optimal feeding program. Initial and final reaction volumes after 81 h were 30 and 45 ml, respectively. The biotransformation was carried out in 2.5 M MOPS, 1 mM MgS04,1 mM TPP with initial pH of 6,5 and initial carboligase activity of 3.82 U mf. The pH adjustment was performed manually using 30% (v/v) H2SO4. 

The project encompassed the comparative characterization of pyruvate decarboxylase from Z. mohilis (PDC) and benzoylformate decarboxylase from P. putida (BED) as well as their optimization for bioorganic synthesis. Both enzymes require thiamine diphosphate (ThDP) and magnesium ions as cofactors. Apart from the decarboxylation of 2-ketoacids, which is the main physiological reaction of these 2-ketoacid decarboxylases, both enzymes show a carboligase site reaction leading to chiral 2-hydroxy ketones (Scheme 2.2.3.1). A well-known example is... 

Pyruvate decarboxylase (PDC, E.C. 4.1.1.1) accepts other substrates besides pyruvate, its natural reactant As early as 1921, Neubergand Hirsch described the reaction of yeast with benzaldehyde and pyruvate to phenylacetylcarbinol (2-keto-3-hydroxy-propylbenzene) (Neuberg, 1921) in a carboligase side reaction which yields ephedrine after reaction with methylamine and catalytic hydrogenation (Figure 7.37). 

Primary hyperoxaluria type I is due to a deficiency of cytosolic a-ketoglutarate-glyoxylate carboligase, which catalyzes the following reaction ... 

Scheme 5 Carboligase side reactions on YPDC and other 2-oxoacid decarboxyiases.

Tartronata-samialdahyda synthase, gfyoxylate carboligase (EC 4.1.1.47) a plant enzyme which converts two molecules of glyoxylate to tartronate semialdehyde and CO2. Hydroxymethyl thiamin pyrophosphate is a reactive intermediate in the reaction. Hie enzyme plays a role in the biosynthesis of carbohydrates from C2 compounds. 

Glyoxalate can synergistically be decarboxylated in the presence of a-ketoglutarate to yield a-hydroxy-jS-ketoadipate. The reaction requires thiamine pyrophosphate, Mn L-glutamate, and NAD. The carboligase... 

Figure 5.9 The use of carbonic anhydrase for the analysis of the reaction product of an enzyme reaction. Hall, Vennesland Ke y (1969) followed the reaction catalysed by glyoxylate carboligase 2CHOCOO" +H - ...

Thiamin diphosphate (ThDP)-dependent carboligases are a class of enzymes involved in a variety of metabolic pathways where they catalyze a broad range of bond cleavage and bond formation reactions between carbon and hydrogen. 

ThDP-dependent carboligases can catalyze acyloin condensations of aliphatic or aromatic donor aldehydes to aliphatic or aromatic acceptors, thus allowing the production of many useftd building blocks (Tables 10.4-10.7). These are the most common reactions catalyzed by ThDP-dependent carboligases such as the pyruvate decarboxylase (PDC) from Saccharomyces cerevisiae (ScPDC), Zymomonas mobilis (ZmPDC), Acetobacter pasteurianus (ApPDC), benzoylformate decarboxylase (BFD) from P. putida (F BFD), and benzaldehyde lyase (BAL) from Psedomonas. fluorescens BidVar 1, which have been recognized as powerful biocatalysts [6,14,48]. 

A possible mechanism is that the pyruvic acid produced during alcoholic fermentation under the influence of a carboxylase yields acetaldehyde in a nascent or specially reactive form, and that the latter then condenses with the benzaldehyde under the asymmetric catalytic effect of carboligase. There is much that still requires elucidation in this reaction, but it should at least be noted that this peculiar new aldol-type reaction cannot be effected by nonenzymic means. 

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