Complete Switch of Reaction Specificity of an Aldolase by Directed Evolution In Vitro: Synthesis of Generic Aliphatic Aldol Products

Abstract

A structure‐guided engineering of fructose‐6‐phosphate aldolase was performed to expand its substrate promiscuity toward aliphatic nucleophiles, that is, unsubstituted alkanones and alkanals. A “smart” combinatorial library was created targeting residues D6, T26, and N28, which form a binding pocket around the nucleophilic carbon atom. Double‐selectivity screening was executed by high‐performance TLC that allowed simultaneous determination of total activity as well as a preference for acetone versus propanal as competing nucleophiles. D6 turned out to be the key residue that enabled activity with non‐hydroxylated nucleophiles. Altogether 25 single‐ and double‐site variants (D6X and D6X/T26X) were discovered that show useful synthetic activity and a varying preference for ketone or aldehyde as the aldol nucleophiles. Remarkably, all of the novel variants had completely lost their native activity for cleavage of fructose 6‐phosphate.

Aldolase‐Catalyzed Asymmetric Synthesis of N‐Heterocycles by Addition of Simple Aliphatic Nucleophiles to Aminoaldehydes

“…As reported previously, the cyclic ketones 1 e and 1 f imposed an E ‐configured FSA K85‐enamine nucleophile complex, contrary to the Z ‐configuration invariably observed with acyclic nucleophiles such as hydroxyethanal, hydroxyacetone or dihydroxyacetone, and other aliphatic ketone nucleophiles . The structural and mechanistic features invariably impose an attack of the nucleophile from its si ‐face to the electrophile, yielding the ( R )‐configuration at C‐α next to the carbonyl group (Scheme ) . The addition of 1 e to 2 b (Table , entry 5), catalyzed by FSA A165G, furnished only diastereomer 15 e indicating a preferential attack at the si ‐face of the electrophilic carbonyl.…”

nanoDSF as screening tool for enzyme libraries and biotechnology development

“…In the study, commercial purified hexokinase from Saccharomyces cerevisiae (HK) was included as received, ketoreductase from L. brevis (KRED Lb ) [39] was used as CFE from recombinant expression and DERA Tm was additionally heat treated and dialysed. Transketolase variant L397F/D399G/H479Q from G. stearothermophilus (TK) [40], fructose-6-phosphate aldolase from E. coli (FSA) [41] and DERA from E. coli (DERA Ec ) were available from current synthetic projects. These enzymes represent distinct folding patterns and come from different organisms, including mesophilic and thermophilic, bacterial and eukaryotic origins.…”

“…DERA Ec was purified to homogeneity by Ni-nitrilotriacetic acid chromatography using its His-tag, lyophilised, stored at À20°C until use and then the lyophilisate was dissolved to 2 gÁL À1 in 50 mM TEA pH 7.5 for use. Transketolase variant L397F/D399G/H479Q from G. stearothermophilus (TK) [40] and fructose-6-phosphate aldolase from E. coli (FSA) [41] were heat treated according to published protocols before lyophilisation, dissolved to 1 and 2 gÁL À1 , respectively, in 50 mM TEA buffer (pH 7.5). Prozomix provided heat treated, dialysed and lyophilised DERA from Thermotoga maritima (DERA Tm ) and lyophilised ketoreductase from L. brevis (KRED Lb ).…”

2-Deoxy-d-ribose-5-phosphate aldolase (DERA): applications and modifications

“…As the number of donors is rather limited, much research is concentrated on finding aldolases with a broader donor scope or aldolases which are specific for yet undescribed donors. A particular success in this area is fructose-6-phosphate aldolase (FSA), an aldolase that accepts dihydroxyacetone as donor but also other ketones and even some aldehydes (Garrabou et al 2009; Junker et al 2018). This donor or nucleophile promiscuity was recently reviewed (Hernandez et al 2018).…”