Benzoylformate decarboxylase

Tsou AY, SC Ransom, JA Gerlt, DD Buechter, PC Babbitt, GL Kenyon (1990) Mandelate pathway of Pseudomonas putida sequence relationships involving mandelate racemase, (5)-mandelate dehydrogenase, and benzoylformate decarboxylase and expression of benzoylformate decarboxylase in Escherichia coli. Biochemistry 29 9856-9862. 

The mandelate pathway in Pseudomonas putida involves successive oxidation to benzoyl formate and benzoate, which is further metabolized via catechol and the 3-ketoadipate pathway (Figure 8.35a) (Hegeman 1966). Both enantiomers of mandelate were degraded through the activity of a mandelate racemase (Hegeman 1966), and the racemase (mdlA) is encoded in an operon that includes the next two enzymes in the pathway—5-mandel-ate dehydrogenase (mdlB) and benzoylformate decarboxylase (mdlC) (Tsou et al. 1990). 

Miiller and co-workers have developed an enantioselective enzymatic crossbenzoin reaction (Table 2) [43, 44], This is the first example of an enantioselective cross-benzoin reaction and takes advantage of the donor-acceptor concept. This transformation is catalyzed by thiamin diphosphate (ThDP) 23 in the presence of benzaldehyde lyase (BAL) or benzoylformate decarboxylase (BFD). Under these enzymatic reaction conditions the donor aldehyde 24 is the one that forms the acyl anion equivalent and subsequently attacks the acceptor aldehyde 25 to provide a variety of a-hydroxyketones 26 in good yield and excellent enantiomeric excesses without contamination of the other cross-benzoin products 27. The authors chose 2-chlorobenzaldehyde 25 as the acceptor because of its inability to form a homodimer under enzymatic reaction conditions. 

BENZENE 1,2-DIOXYGENASE PHTHALATE DIOXYGENASE BENZOATE-CoA LIGASE BENZOATE 1,2-DI OXYGENASE BENZOYLFORMATE DECARBOXYLASE BERBERINE BRIDGE ENZYME BETA (j8)... 

Thiamin-dependent enzymes, ACETOLACTATE SYNTHASE BENZOYLFORMATE DECARBOXYLASE BRANCHED-CHAIN a-KETO ACID DEHYDROGENASE COMPLEX... 

Directed Evolution to Increase the Substrate Range of Benzoylformate Decarboxylase from Pseudomonas putida... 

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). 

Scheme 2.2.1.1 Mandelate pathway benzoylformate decarboxylase (BFD), encoded by the gene md/C, catalyzes the conversion of benzoylformate 3 to benzaldehyde 4 and carbon dioxide.

In previous studies on benzoylformate decarboxylase (BFD) from Pseudomonas putida, the enzyme was expressed using the pKK233-3 vector system and E. coli SG13009 as the expression host. Here, expression was controlled by two plasmids. 

Table 2.2.1.3 Synthesis of (R)-benzoin catalyzed by benzoylformate decarboxylase variants.

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 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... 

M. S. Hasson, A. Muscate, M. J. McLeish, L. S. Polovnikova, J. A. Gerlt, G. L. Kenyon, G. A. Petsko, D. Ringe, The crystal structure of benzoylformate decarboxylase at 1.6 A resolution, diversity of catalytic residues in thiamin diphosphate-dependent enzymes. Biochemistry 1998, 37, 9918-9930. 

Grotzinger, A. S. Demir, M. Pohl, Benzoylformate decarboxylase from Pseudomonas putida as stable catalyst for the synthesis of chiral 2-hydroxy ketones. Chem. Eur.J. 2000, 6, 1483-1495. 

M. Pohl, Exchanging the substrate specificities of pyruvate decarboxylase from Zymomonas mohilis and benzoylformate decarboxylase from Pseudomonas putida. Protein Eng. Des. 

B. Lingen, J. Grotzinger, D. Kolter, M. R. Kula, M. Pohl, Improving the carboligase activity of benzoylformate decarboxylase from Pseudomonas putida by a combination of directed evolution and site-directed mutagenesis. Protein Eng. 2002, 15, 585-593. 

LB-ADH was used for the synthesis of vic-diols. Starting from benzaldehyde and acetaldehyde, (lS,2S)-l-phenylpropane-l,2-diol (de = 98%) and (lS,2R)-l-phenyl-propane-l,2-diol (de = 99%), respectively, could be produced in a stereoselective two-step enzymatic synthesis using benzaldehyde lyase (BAL) and accordingly benzoylformate decarboxylase (BFD) as well as LB-ADH [8] (Scheme 2.2.4.3). 

The potential of benzoylformate decarboxylase (BFD, E.C. 4.1.1.7) to catalyze C-C bond formation was first reported by Wilcocks at al. using crude extracts of Pseudomonas putidsL [50]. They observed the formation of (S)-2-hydroxy-l-phenylpro-panone (S)-2-HPP when benzoyl formate was decarboxylated in the presence of acetaldehyde. Advantageously, aldehydes - without a previous decarboxylation step - can be used instead of the corresponding more expensive a-keto acids [51]. We could show that BFD is able to bind a broad range of different aromatic, heteroaromatic, and even cyclic aliphatic and conjugated olefinic aldehydes to ThDP before ligation to acetaldehyde or other aldehydes (Table 2.2.7.3) [52]. 

In a first reactor, where benzoylformate decarboxylase (BFD) is retained, benz-aldehyde and acetaldehyde are coupled to yield (S)-hydroxy-l-phenylpropanone. This hydroxy ketone is then reduced to the corresponding diol in a second reactor by an alcohol dehydrogenase (ADH). Regeneration of the necessary cofactor is achieved by formate dehydrogenase (FDH) or by other methods. To avoid additional consumption of redox equivalents by unselective reduction of residual starting material from the first reactor, the volatile aldehydes are removed via an inline stripping module between the two membrane reactors. In this setup the diol was produced with high optical purity (ee, de > 90%) at an overall space-time yield of 32 g L d . ... 

Fig. 3.1.5 Cascade of membrane reactors with isolated enzymes (BFD benzoylformate decarboxylase ADH alcohol dehydrogenase FDH formate dehydrogenase).

By 1998, X-ray structures had been determined for four thiamin diphosphate-dependent enzymes (1) a bacterial pyruvate oxidase,119120 (2) yeast and bacterial pyruvate decarboxylases,121 122c (3) transketolase,110123124 and (4) benzoylformate decarboxylase.1243 Tire reactions catalyzed by these enzymes are all quite different, as are the sequences of the proteins. However, the thiamin diphosphate is bound in a similar way in all of them. 

Results of a kinetic study of enamine formation by C(2a)-proton abstraction from 2-benzylthiazolium salts (88) have implications for mechanistic studies of the thiamin diphosphate-dependent enzymes which feature protonation of the enamine/C(2a)-carbanion.151 The primary isotope effect for deprotonation of (88a) is kiw/kro = 4-6 and the values estimated for C(2a)—H pXa are 15.0-15.5 and 15.7 for (88a) and (88b), respectively. A minimum effective molarity of 4500 M has been estimated for reprotonation of the enamine (89b) derived from (88b) by benzoylformate decarboxylase. Directed aromatic metallation reactions have been reviewed.152... 

Benzoylformate decarboxylase (BFD), a TPP-dependent enzyme, features a high degree of sequence similarity with a whole family of other TPP-dependent decarboxylases, among them pyruvate decarboxylase (PDC), one of the most important enzymes in metabolism. 

As an obvious extension of the benzoin reaction, the cross-coupling of aldehydes or of aldehydes and ketones was first achieved with the thiamine-dependent enzyme benzoylformate decarboxylase. This linked a variety of mostly aromatic aldehydes to acetaldehyde to form the corresponding a-hydroxy ketones, both chemo- and stereoselectively [31]. Synthetic thiazolium salts, developed by Stetter and co-workers and similar to thiamine itself [32], have been successfully used by Suzuki et al. for a diastereoselective intramolecular crossed aldehyde-ketone benzoin reaction during the course of an elegant natural product synthesis [33], Stereocontrol was exerted by pre-existing stereocenters in the specific substrates, the catalysts being achiral. 

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