S‐Selective Mixed Carboligation by Structure‐Based Design of the Pyruvate Decarboxylase from Acetobacter pasteurianus

Abstract

The thiamine diphosphate (ThDP)‐dependent pyruvate decarboxylase from Acetobacter pasteurianus (ApPDC) catalyzes the carboligation of aldehydes that yields (R)‐2‐hydroxy ketones with high chemoselectivity in mixed carboligations of aliphatic donor and aromatic acceptor aldehydes. On the basis of the crystal structure of ApPDC, which was determined to a resolution of 2.75 Å, and biochemical data, we mapped the active site. This enabled us to design variants with tailor‐made catalytic activities by modifications of the residues E469 and W388. Although the exchange of W388 by smaller amino acids yields variants with higher carboligase activity due to an increased access to the active site, the exchange of E469 to glycine opens the so‐called S‐pocket in ApPDC for aromatic aldehydes and thus alters the stereoselectivity. The variant ApPDC‐E469G provides access to (S)‐phenylacetylcarbinol derivatives by enzymatic carboligation with a good stereoselectivity of up to 89 % enantiomeric excess. The variant nicely complements the toolbox of ThDP‐dependent enzymes, which now gives access to all stereo‐ and regioisomers of the asymmetric aliphatic–aromatic cross‐benzoin‐like condensation. We prove that optimal stabilization of both aldehydes in the active site is essential to gain high yields and high selectivities.

A standard numbering scheme for thiamine diphosphate-dependent decarboxylases

“…With the exception of a few functionally relevant residues that have been identified by comparing sequences and structures of homologous proteins or by mutation experiments, the molecular basis of this biochemical diversity is still unknown. Variants have been developed by rational design and by directed evolution, in order to improve the activity of members of this enzyme family [16,20,21] or to alter substrate specificity [22-28] or stereoselectivity [29-31]. Some functionally relevant amino acids are located in the active site, mediating substrate binding [3], are involved in the activation of ThDP [28] or steer stereoselectivity [29-31], e.g.…”

“…Variants have been developed by rational design and by directed evolution, in order to improve the activity of members of this enzyme family [16,20,21] or to alter substrate specificity [22-28] or stereoselectivity [29-31]. Some functionally relevant amino acids are located in the active site, mediating substrate binding [3], are involved in the activation of ThDP [28] or steer stereoselectivity [29-31], e.g. the S -pocket as part of the acceptor binding site, which has been shown to contribute to the stereoselectivity of several members of the decarboxylase superfamily [29-31].…”

“…Some functionally relevant amino acids are located in the active site, mediating substrate binding [3], are involved in the activation of ThDP [28] or steer stereoselectivity [29-31], e.g. the S -pocket as part of the acceptor binding site, which has been shown to contribute to the stereoselectivity of several members of the decarboxylase superfamily [29-31]. However, due to this complexity, combining results yielded from different variants of different protein families, consolidating results on the function of specific residues and comparing results from different research groups is unfortunately not a straightforward process.…”

Mechanistic and Structural Insight to an Evolved Benzoylformate Decarboxylase with Enhanced Pyruvate Decarboxylase Activity

“…The archetypal member of this group is pyruvate decarboxylase (PDC), which catalyzes the nonoxidative decarboxylation of pyruvate to yield acetaldehyde and carbon dioxide, a reaction critical to the fermentation pathway of several yeast and bacteria [2]. X-ray structures of PDCs from a variety of species show that, in addition to ThDP, the active site contains two ionizable acidic residues as well as two contiguous histidine residues that are located on an ordered loop [3][4][5][6]. The latter has been termed the HH-motif, and mutagenesis and kinetic studies have revealed that both histidines play significant roles in catalysis [7][8][9][10].…”

“…Catalysts 2016, 6,190 3 of 16 As with many ThDP-dependent decarboxylases, BFDC and PDC are both are capable of stereoselective carboligation reactions leading to chiral α-hydroxy ketones [26]. These have been used as versatile building blocks in the pharmaceutical (and other) industries [26][27][28].…”