Benzoylformate decarboxylase (BFD) from Pseudomonas putida is an exceptional thiamin diphosphate-dependent enzyme, as it catalyzes the formation of (S)-2-hydroxy-1-phenylpropan-1-one from benzaldehyde and acetaldehyde. This is the only currently known S-selective reaction (92 % ee) catalyzed by this otherwise R-selective class of enzymes. Here we describe the molecular basis of the introduction of S selectivity into ThDP-dependent decarboxylases. By shaping the active site of BFD through the use of rational protein design, structural analysis, and molecular modeling, optimal steric stabilization of the acceptor aldehyde in a structural element called the S pocket was identified as the predominant interaction for adjusting stereoselectivity. Our studies revealed Leu461 as a hot spot for stereoselectivity in BFD. Exchange to alanine and glycine resulted in variants that catalyze the S-stereoselective addition of larger acceptor aldehydes, such as propanal with benzaldehyde and its derivatives-a reaction not catalyzed by the wild-type enzyme. Crystal structure analysis of the variant BFDL461A supports the modeling studies.

Pyruvate decarboxylase (PDC) catalyzes the decarboxylation of pyruvate into acetaldehyde and CO(2) and requires the cofactors thiamin diphosphate and Mg(2+) for activity. Owing to its catalytic promiscuity and relaxed substrate specificity, PDC catalyzes carboligation side reactions and is exploited for the asymmetric synthesis of 2-hydroxy ketones such as (R)-phenylacetyl carbinol, the precursor of (-)-ephedrine. Although PDC variants with enhanced carboligation efficiency were generated in the past, the native reaction, i.e., formation of aldehydes, is heavily favored over carboligation side reactions in all these biocatalysts. We characterized an active site variant (Glu473Gln) in which partitioning between aldehyde release versus carboligation is inverted with an up to 100-fold preference for the latter pathway. Due to a defective protonation of the central carbanion/enamine intermediate, substrate turnover stalls at this catalytic stage and addition of external aldehydes leads to quantitative and enantioselective formation of 2-hydroxy ketones as shown for (R)-phenylacetyl carbinol, which is afforded with unmatched yields, rates, and purity. This protein variant thus constitutes an example for the rational design of biocatalysts with greatly enhanced accidental catalytic promiscuity by selective blockage of the native reaction and accumulation of reactive intermediates under steady-state turnover conditions.

The thiamine diphosphate-dependent, branched-chain 2-keto acid decarboxylase from Lactococcus lactis sup. cremoris B1157 (KdcA) is a new valuable enzyme for the synthesis of chiral 2-hydroxy ketones. The gene was cloned and the enzyme was expressed as an N-terminal hexahistidine fusion protein in Escherichia coli. It has a broad substrate range for the decarboxylation reaction including linear and branched-chain aliphatic and aromatic keto acids as well as phenyl pyruvate and indole-3pyruvate. The dimeric structure of recombinant KdcA is in contrast to the tetrameric structure of other 2-keto acid decarboxylases. The enzyme is stable between pH 5 and 7 with a pH optimum of pH 6-7 for the decarboxylation reaction. While KdcA is sufficiently stable up to 40 8C it rapidly looses activity at higher temperatures. In this work the carboligase activity of KdcA is demonstrated for the first time. The enzyme shows an exceptionally broad substrate range and, most strikingly, it catalyzes the carboligation of different aromatic aldehydes as well as CH-acidic aldehydes such as phenylacetaldehyde and indole-3-acetaldehyde with aliphatic aldehydes such as acetaldehyde, propanal, and cyclopropanecarbaldehyde, yielding chiral 2-hydroxy ketones in high enantiomeric excess. Noteworthy, the donor-acceptor selectivity is strongly influenced by the nature of the respective substrate combination.

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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...