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Engineering an aldolase for catalysis of rare-to-nature C-C bond formations

The enzyme 2-deoxy-D-ribose-5-phosphate aldolase (DERA) was redesigned into a variant capable of catalysing rare-to-nature C-C bond-forming Michael addition reactions, yielding chiral precursors of pharmaceutically active gamma-aminobutyric acid analogs. X-ray diffraction analysis enabled the three-dimensional structural characterisation of a DERA variant, elucidating how the engineered mutations changed the enzyme from an aldolase into a Michaelase .

The class I aldolase 2-deoxy-D-ribose-5-phosphate aldolase (DERA) from Escherichia coli catalyses the reversible aldol addition of acetaldehyde and D-glyceraldehyde-3-phosphate to give 2-deoxy-D-ribose- 5-phosphate. This reaction, which is well understood [1], proceeds via the formation of a Schiff-base intermediate with a conserved active site lysine (Lys167, Figure 38a). Over the past 20 years, DERA has found extensive application as a biocatalyst owing to its versatility in catalysing asymmetric C-C bond-forming aldol reactions [2] and to its susceptibility towards expanding its substrate scope by protein engineering [3,4].

This study investigated whether DERA may also catalyse novel, rare-to-nature carboligation reactions, so as to further increase the potential of this enzyme for useful biotechnological applications. Surprisingly, the wild-type enzyme displayed very weak promiscuous activity in a Michael addition reaction with the unnatural substrates cinnamaldehyde and nitromethane, yielding the product 4-nitro-3-phenyl-butanal (a γ-nitroaldehyde, Figure 38b). Using a highly effective directed-evolution campaign, the wildtype enzyme was subsequently redesigned into

a variant with 12 amino-acid substitutions, which lost the native retro-aldolase activity, instead displaying an impressive 190-fold enhancement in catalytic activity for Michael additions. This new DERA variant (DERA- MA) was remarkably versatile in accepting a variety of cinnamaldehyde derivatives as substrates, achieving high conversions (>95%), excellent product enantiopurity (e.r. = 99:1) and good product yields at a semi- preparative scale. Notably, some of the γ-nitroaldehyde products produced by DERA-MA are valuable synthetic precursors for the synthesis of pharmaceutically active γ-aminobutyric acid (GABA) derivatives.

To reveal the mechanism by which DERA-MA catalyses the Michael addition reaction, and to rationalise the effect of the mutations, high-resolution crystal structures were determined of DERA-MA in a substrate-free state and in a covalent reaction intermediate state obtained by briefly soaking crystals with the substrate cinnamaldehyde (Figure 39). X-ray diffraction data were recorded at beamline ID30A-1. The overall structure of DERA-MA is highly similar to that of the wildtype enzyme and displays the typical (α/β)8 TIM-barrel fold. Ten of the 12 mutations are located in loops that surround the active site pocket. Two of these loops display a significant change in backbone conformation compared to the wildtype structure, exposing a new apolar subpocket for binding the phenyl-moiety of cinnamaldehyde, while negatively affecting binding of the natural substrate D-glyceraldehyde-3-phosphate. The 1.9 Å electron density map obtained for the cinnamaldehyde- soaked crystal allowed a clear characterisation of the Schiff-base intermediate formed between the substrate and Lys167. The DERA-MA crystal structure of this Schiff- base complex reveals that the re-face of carbon C3 is easily accessible for a nucleophilic attack by nitromethane, while its si-face is blocked due to contacts with active site residues, thus explaining the stereochemistry of the overall reaction. The crystallographic results further support the

Fig. 38: a) Natural aldol reaction catalysed by wildtype DERA. Formation of a covalent enamine intermediate between acetaldehyde and Lys167 is followed by a nucleophilic attack on D-glyceraldehyde-3- phosphate. b) Proposed reaction steps for the Michael reaction catalysed by DERA-MA. Catalysis proceeds via a covalent iminium intermediate formed between cinnamaldehyde and Lys167, followed by a nucleophilic attack of nitromethane.