Branched‐Chain Keto Acid Decarboxylase from <i>Lactococcus lactis</i> (KdcA), a Valuable Thiamine Diphosphate‐Dependent Enzyme for Asymmetric CC Bond Formation

Gocke, Dörte; Nguyen, Cong Luan; Pohl, Martina; Stillger, Thomas; Walter, Lydia; Müller, Michael

doi:10.1002/adsc.200700057

Cited by 50 publications

(65 citation statements)

References 26 publications

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“…Consequently, it is one of the enzymes in our study with the broadest product range, including HPP, PAC and acetoin 12. Only the bulky benzoin, derived from the ligation of two molecules of benzaldehyde, is not accessible.…”

Section: Resultsmentioning

confidence: 99%

“…Here the acetaldehyde also has to be arranged in an antiparallel manner to the donor aldehyde. The S -pocket in Ll KdcA is blocked by amino acid side chains and thus not accessible according to the available crystal structure 12. However, also in a buffered system the enzyme is able to produce small amounts of the S -product, yielding an ee of 95.6% for ( R )-HPP (Figure 4, Figure 8).…”

Section: Discussionmentioning

confidence: 99%

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Influence of Organic Solvents on Enzymatic Asymmetric Carboligations

et al. 2012

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The asymmetric mixed carboligation of aldehydes with thiamine diphosphate (ThDP)-dependent enzymes is an excellent example where activity as well as changes in chemo- and stereoselectivity can be followed sensitively. To elucidate the influence of organic additives in enzymatic carboligation reactions of mixed 2-hydroxy ketones, we present a comparative study of six ThDP-dependent enzymes in 13 water-miscible organic solvents under equivalent reaction conditions. The influence of the additives on the stereoselectivity is most pronounced and follows a general trend. If the enzyme stereoselectivity in aqueous buffer is already >99.9% ee, none of the solvents reduces this high selectivity. In contrast, both stereoselectivity and chemoselectivity are strongly influenced if the enzyme is rather unselective in aqueous buffer. For the S-selective enzyme with the largest active site, we were able to prove a general correlation of the solvent-excluded volume of the additives with the effect on selectivity changes: the smaller the organic solvent molecule, the higher the impact of this additive. Further, a correlation to log P of the additives on selectivity was detected if two additives have almost the same solvent-excluded volume. The observed results are discussed in terms of structural, biochemical and energetic effects. This work demonstrates the potential of medium engineering as a powerful additional tool for varying enzyme selectivity and thus engineering the product range of biotransformations. It further demonstrates that the use of cosolvents should be carefully planned, as the solvents may compete with the substrate(s) for binding sites in the enzyme active site.

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Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Influence of Organic Solvents on Enzymatic Asymmetric Carboligations

et al. 2012

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“…The decrease in NADH concentration was monitored photometrically (Gocke et al 2007). According to these experiments, no pyruvate decarboxylase activity could be detected.…”

Section: 2-carboligation Activitymentioning

confidence: 99%

Asymmetric Stetter reactions catalyzed by thiamine diphosphate-dependent enzymes

Kasparyan

Richter

Dresen

et al. 2014

Appl Microbiol Biotechnol

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The intermolecular asymmetric Stetter reaction is an almost unexplored transformation for biocatalysts. Previously reported thiamine diphosphate (ThDP)-dependent PigD from Serratia marcescens is the first enzyme identified to catalyze the Stetter reaction of α,β-unsaturated ketones (Michael acceptor substrates) and α-keto acids. PigD is involved in the biosynthesis of the potent cytotoxic agent prodigiosin. Here, we describe the investigation of two new ThDP-dependent enzymes, SeAAS from Saccharopolyspora erythraea and HapD from Hahella chejuensis. Both show a high degree of homology to the amino acid sequence of PigD (39 and 51 %, respectively). The new enzymes were heterologously overproduced in Escherichia coli, and the yield of soluble protein was enhanced by co-expression of the chaperone genes groEL/ES. SeAAS and HapD catalyze intermolecular Stetter reactions in vitro with high enantioselectivity. The enzymes possess a characteristic substrate range with respect to Michael acceptor substrates. This provides support for a new type of ThDP-dependent enzymatic activity, which is abundant in various species and not restricted to prodigiosin biosynthesis in different strains. Moreover, PigD, SeAAS, and HapD are also able to catalyze asymmetric carbon-carbon bond formation reactions of aldehydes and α-keto acids, resulting in 2-hydroxy ketones.

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“…Nevertheless, influence of substrate ratio on donor/acceptor properties are well known for other ThDP-dependent enzymes. [22] With BFDwt as a catalyst cyclohex-1-enecarbaldehyde (1e) was converted to (S)-3e with moderate yield and good enantioselectivity (entry 12).[23] Moreover, hexenal (1g) and octenal (1h) were also donor substrates for BFDwt in the presence of acetaldehyde (entry 16 and 19). The only substrate for BFD-H281A in the ligation with acetaldehyde was hexenal (1g) with moderate conversion to give 3g with poor enantioselectivity (entry 17).…”

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α,β‐Unsaturated Aldehydes as Substrates for Asymmetric CC Bond Forming Reactions with Thiamin Diphosphate (ThDP)‐Dependent Enzymes

et al. 2008

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The enzymes benzaldehyde lyase (BAL) from Pseudomonas fluorescens, benzoylformate decarboxylase (BFD) from Pseudomonas putida and pyruvate decarboxylase (PDC) from Saccharomyces cerevisiae provide different C À C bond forming possibilities of a,b-unsaturated aldehydes with aliphatic and aromatic aldehydes. Structure elucidation and determination of the absolute configuration of the products, which were obtained with high regio-and stereoselectivity were carried out. Selective 1,2-reactivity with yields of 75% and > 98% ee, for one single isomer (A) were obtained, by choosing the suitable enzyme in combination with the appropriate substrates. By varying enzymes or substrates the regioisomeric hydroxy ketones C, with up to > 99% ee, can be obtained. The application of these new chiral building blocks in the synthesis of natural products or biological active substances is considerably facilitated by applying the different ThDP-dependent enzymes as catalysts.Abbreviations: BAL, benzaldehyde lyase; BFD, benzoylformate decarboxylase; PDC, pyruvate decarboxylase; His, hexahistidine; 2-HPP, 2-hydroxy-1-phenylpropan-1-one; PAC, phenylacetylcarbinol; NTA, nitrilotriacetic acid; ThDP, thiamin diphosphate; wt, wild-type.Keywords: benzaldehyde lyase; benzoylformate decarboxylase; biocatalysts; pyruvate decarboxylase; stereochemistry IntroductionThiamin diphosphate (ThDP)-dependent enzymes are well-known to catalyse a broad range of asymmetric reactions.[1] Enzymes of this class like benzaldehyde lyase (BAL) from Pseudomonas fluorescens, [2] benzoylformate decarboxylase (BFD) from Pseudomonas putida [3] and pyruvate decarboxylase (PDC) from Saccharomyces cerevisiae [4] enable various modes of C À C bond forming reactions. Besides the physiological reactivity, several of these enzymes can catalyse, for example, the C À C bond formation between two aldehydes, giving chiral 2-hydroxy ketones with high enantioselectivity. The reactions of aromatic aldehydes as donor and aliphatic aldehydes as acceptor give rise to 2-hydroxy-1-phenyl-1-propanone derivatives with S absolute configuration in the case of BFD catalysis [5] and R for BAL.[6] If aromatic aldehydes are employed as donor and acceptor, (R)-benzoins are optained with both enzymes. [7,8] (R)-Phenylacetylcarbinol is formed in the PDC-catalysed reaction of decarboxylated pyruvate with benzaldehyde as acceptor aldehyde. [4,9] Here we describe the use of a,b-unsaturated aldehydes as substrates for C À C bond ligation in BAL-, BFD-and PDC-catalysed reactions. These substrates might act as donor and as acceptor for the 1,2-(A, C) and the 1,4-addition (B, D) (Scheme 1). Adv. Synth. Catal. 2008, 350, 759 -771 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim 759 FULL PAPERSIn reactions according to Equation (1) the a,b-unsaturated aldehyde is bound to ThDP and reacts as donor resulting in the formation of hydroxy ketone A or hydroxy enone B. In reactions according to Equation (2) the a,b-unsaturated aldehyde is attacked by an "active aldehyde" and reacts as acceptor resulti...

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Branched‐Chain Keto Acid Decarboxylase from Lactococcus lactis (KdcA), a Valuable Thiamine Diphosphate‐Dependent Enzyme for Asymmetric CC Bond Formation

Cited by 50 publications

References 26 publications

Influence of Organic Solvents on Enzymatic Asymmetric Carboligations

Influence of Organic Solvents on Enzymatic Asymmetric Carboligations

Asymmetric Stetter reactions catalyzed by thiamine diphosphate-dependent enzymes

α,β‐Unsaturated Aldehydes as Substrates for Asymmetric CC Bond Forming Reactions with Thiamin Diphosphate (ThDP)‐Dependent Enzymes

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