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.

). An ipdC-knockout mutant was found to produce only 10% (wt/vol) of the wild-type IAA production level. In this study, the encoded enzyme is characterized via a biochemical and phylogenetic analysis. Therefore, the recombinant enzyme was expressed and purified via heterologous overexpression in Escherichia coli and subsequent affinity chromatography. The molecular mass of the holoenzyme was determined by size-exclusion chromatography, suggesting a tetrameric structure, which is typical for 2-keto acid decarboxylases. The enzyme shows the highest k cat value for phenylpyruvate. Comparing values for the specificity constant k cat /K m , indole-3-pyruvate is converted 10-fold less efficiently, while no activity could be detected with benzoylformate. The enzyme shows pronounced substrate activation with indole-3-pyruvate and some other aromatic substrates, while for phenylpyruvate it appears to obey classical Michaelis-Menten kinetics. Based on these data, we propose a reclassification of the ipdC gene product of A. brasilense as a phenylpyruvate decarboxylase (EC 4.1

Thiamin diphosphate-dependent enzymes participate in numerous biosynthetic pathways and catalyse a broad range of reactions, mainly involving the cleavage and formation of C-C bonds. For example, they catalyse the nonoxidative and oxidative decarboxylation of 2-keto acids, produce 2-hydroxy ketones and transfer activated aldehydes to a variety of acceptors. Moreover, they can also catalyse C-N, C-O and C-S bond formation. Because of their substrate spectra and different stereospecificity, these enzymes extend the synthetic potential for asymmetric carboligations appreciably. Different strategies have been developed to identify new members of this promiscuous enzyme class and the reactions they catalyse. This enabled us to introduce solutions for longstanding synthetic problems, such as asymmetric cross-benzoin condensation. Moreover, through a combination of protein structure analysis, enzyme and substrate engineering, and screening methods we explored additional stereochemical routes that have not been described previously for any of these interesting enzymes.

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.

The thiamin diphosphate (ThDP) dependent branched-chain keto acid decarboxylase (KdcA) from Lactococcus lactis catalyzes the decarboxylation of 3-methyl-2-oxobutanoic acid to 3-methylpropanal (isobutyraldehyde) and CO2. The enzyme is also able to catalyze carboligation reactions with an exceptionally broad substrate range, a feature that makes KdcA a potentially valuable biocatalyst for C-C bond formation, in particular for the enzymatic synthesis of diversely substituted 2-hydroxyketones with high enantioselectivity. The crystal structures of recombinant holo-KdcA and of a complex with an inhibitory ThDP analogue mimicking a reaction intermediate have been determined to resolutions of 1.6 and 1.8 A, respectively. KdcA shows the fold and cofactor-protein interactions typical of thiamin-dependent enzymes. In contrast to the tetrameric assembly displayed by most other ThDP-dependent decarboxylases of known structure, KdcA is a homodimer. The crystal structures provide insights into the structural basis of substrate selectivity and stereoselectivity of the enzyme and thus are suitable as a framework for the redesign of the substrate profile in carboligation reactions.