ThDP-dependent enzymes

Benzoylformate decarboxylase (BFD EC 4.1.1.7) belongs to the class of thiamine diphosphate (ThDP)-dependent enzymes. ThDP is the cofactor for a large number of enzymes, including pyruvate decarboxylase (PDC), benzaldehyde lyase (BAL), cyclohexane-1,2-dione hydrolase (CDH), acetohydroxyacid synthase (AHAS), and (lR,6] )-2-succinyl-6-hydroxy-2,4-cyclohexadiene-l-carboxylate synthase (SHCHC), which all catalyze the cleavage and formation of C-C bonds [1]. The underlying catalytic mechanism is summarized elsewhere [2] (see also Chapter 2.2.3). 

Therefore, ThDP-dependent enzymes include the potential of both making and breaking of C-C bonds [1]. All enzymes have in common a ThDP-bound active aldehyde intermediate formed either by decarboxylation or by transfer from a suitable donor compound (e.g., from xylulose-5-phosphate by transketolase). Some of the decarboxylating enzymes also catalyze interesting side-reactions where two aldehydes are joined, resulting in so-called acyloin condensations [1,... 

In the framework of SFB380, two projects dealt extensively with acyloin-con-densing ThDP-dependent enzymes such as pyruvate decarboxylase (PDC), ben-zoylformate decarboxylase (BFD), or benzaldehyde lyase (BAL) (see Chapters 2.2.3 and 2.2.7). Another ThDP-dependent decarboxylase, phosphonopyruvate decarboxylase (PPD) from Streptomyces viridochromogenes, became available only recently and was studied in project B21. We wanted to find out whether this PDC-related enzyme could be a valuable tool in the provision of acyloin condensations involving C-P bonds (see Section 2.2.2.23). 

C-P bonds are present in a range of natural compounds, for example, antibiotics. Through a reaction of PEP mutase, phosphoenolpyruvate is converted to phosphonopyruvate, which is the precursor of all natural phosphono compounds. The ThDP-dependent enzyme phosphonopyruvate decarboxylase (PPD) has been discovered in several Streptomycetes and other bacteria [24] its reaction is depicted in Scheme 2.2.2.3. 

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. 

The selective donor-acceptor concept can be transferred to other ThDP-dependent enzymes. For example, enantiopure mixed benzoins were obtained when 2-chlorobenzaldehyde reacted with a variety of selective donor aldehydes in the presence of BAL [67]. By performing various cross-benzoin condensation reactions with this enzyme, not only new selective donors but also additional aldehydes reacting selectively as acceptors, such as 2-iodobenzaldehyde or 2,6-difluorobenzaldehyde, could be identified. Again all the mixed benzoins generated exhibited an R-configuration and were obtained with high to excellent enantiomeric excesses [69]. 

From the results mentioned above, it can be proposed that a detailed investigation of different ThDP-dependent enzymes using substrate and protein engineering, with the focus on new transformations, will open new perspectives in catalytic... 

Until now only TK [49a], PDC [49b], BFD, BAL [72], and mutants thereof have been investigated systematically with regard to preparative transformations. Numerous other ThDP-dependent enzymes are capable of catalyzing different asymmetric reactions as well [73]. 

For some reviews of ThDP-dependent enzymes with regard to asymmetric synthesis see a) G. A. Sprenger, M. Pohl,/. Mol. Catal. E Enzym. 1999, 6, 145-159 b) U. Schorken, G. A. 

As noted earlier, the conversion of benzoylformic acid to benzaldehyde is catalyzed by the thiamin diphosphate (ThDP)-dependent enzyme BFD. The proposed catalytic mechanism proceeds through two covalent... 

The reaction cycle is started with the activation of ThDP by the enzyme. For this common step in all ThDP-dependent enzymes, a stepwise and a concerted... 

The 3D-structure of the oc4 isoenzyme of PDC from S. uvarum (PDCS.u.) was reported by Dyda and co-workers in 1993 [55], a short time after the publication of the crystal structures of two other ThDP-dependent enzymes, the... 

The electronic spectroscopic data collected both on the model enamines and on the PDC-bound ones leave little doubt that all of these enamines are planar, highly conjugated structures. Therefore, invoking a pyramidal C2a atom in the enamine intermediates no longer offers a valid hypothesis, at least on PDC, and likely not on other ThDP-dependent enzymes either. 

A second class of ThDP-dependent enzymes, that perform the biological equivalent of the benzoin condensation reaction, interconvert sugar phosphates of different chain lengths. In the pentose shunt transketolase catalyzes the reaction shown in Scheme 10,... 

The deprotonation and addition of a base to thiazolium salts are combined to produce an acyl carbanion equivalent (an active aldehyde) [363, 364], which is known to play an essential role in catalysis of the thiamine diphosphate (ThDP) coenzyme [365, 366]. The active aldehyde in ThDP dependent enzymes has the ability to mediate an efScient electron transfer to various physiological electron acceptors, such as lipoamide in pyruvate dehydrogenase multienzyme complex [367], flavin adenine dinucleotide (FAD) in pyruvate oxidase [368] and Fc4S4 cluster in pyruvate ferredoxin oxidoreductase [369]. 

Pyruvate oxidase (Pyox) is a FAD- and thiamine diphosphate (ThDP)-dependent enzyme that catalyzes the reaction of pyruvate to give acetyl phosphate or vice versa (see Fig. 15). If used in the oxidative way, it can be activated and reactivated under nonaerobic conditions using ferrocene mediators. Kinetic parameters of the indirect electrochemical process using the enzyme incorporated into a biomimetic supported bilayer at a gold electrode have been reported [142]. Similarly, FAD-dependent amino oxidases may also be applied. 

This example demonstrates that non-natural transformation of known enzymes is an extremely helpful tool for organic chemists [21]. Recently, other ThDP-dependent enzymes have been used for the asymmetric synthesis of many different 2-hydroxy ketones (Scheme 4.3) [22, 23]. 

In these reactions, the C2-atom of ThDP must be deprotonated to allo v this atom to attack the carbonyl carbon of the different substrates. In all ThDP-dependent enzymes this nucleophilic attack of the deprotonated C2-atom of the coenzyme on the substrates results in the formation of a covalent adduct at the C2-atom of the thiazolium ring of the cofactor (Ila and Ilb in Scheme 16.1). This reaction requires protonation of the carbonyl oxygen of the substrate and sterical orientation of the substituents. In the next step during catalysis either CO2, as in the case of decarboxylating enzymes, or an aldo sugar, as in the case of transketo-lase, is eliminated, accompanied by the formation of an a-carbanion/enamine intermediate (Ilia and Illb in Scheme 16.1). Dependent on the enzyme this intermediate reacts either by elimination of an aldehyde, such as in pyruvate decarboxylase, or with a second substrate, such as in transketolase and acetohydroxyacid synthase. In these reaction steps proton transfer reactions are involved. Furthermore, the a-carbanion/enamine intermediate (Ilia in Scheme 16.1) can be oxidized in enzymes containing a second cofactor, such as in the a-ketoacid dehydrogenases and pyruvate oxidases. In principal, this oxidation reaction corresponds to a hydride transfer reaction. 

Whereas the mechanism of the C2-H deprotonation of ThDP has been shown to be identical in all ThDP-dependent enzymes investigated, the following steps in catalysis of the different enzymes require different protonation and deprotonation reactions of the intermediates formed along the process. In order to identify side chains involved in proton transfer steps, the distribution of reaction intermediates during catalysis of any wild type enzyme can be compared with that of active site mutant enzymes. Rate constants for single steps in catalysis can be calculated from... 

LThDP) was among the first examples of a covalently bound predecarboxylation reaction intermediate analog in any ThDP-dependent enzyme. ... 

Several early structural models of AHAS were proffered based on homology to other known ThDP-dependent enzymes, such as POX from Lactobacillus planta-rum, and carefully planned site-directed mutagenesis studies. Many of the features of the early models were borne out by the first X-ray crystal structure at 2.6-A resolution of the dimeric catalytic subunit of AHAS from S. cerevisiae [15,... 

The broad synthetic potential ThDP-dependent enzymes for asymmetric C-C bond formation is by far not fully exploited with the acyloin- and benzoin-condensations discussed above. On the one hand, novel branched-chain a-keto-acid decarboxylases favorably extend the limited substrate tolerance of traditirnial enzymes, such as PDC, by accepting sterically hindered a-ketoacids as dcmors [1511], On the other hand, the acceptor range may be significantly widened by using carlxMiyl compounds other than aldehydes Thus, ketones, a-ketoacids and even CO2 lead to novel types of products (Scheme 2.203). 

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