Current state of and need for enzyme engineering of 2-deoxy-D-ribose 5-phosphate aldolases and its impact

Abstract

Deoxyribose-5-phosphate aldolases (DERAs, EC 4.1.2.4) are acetaldehyde-dependent, Class I aldolases catalyzing in nature a reversible aldol reaction between an acetaldehyde donor (C2 compound) and glyceraldehyde-3-phosphate acceptor (C3 compound, C3P) to generate deoxyribose-5-phosphate (C5 compound, DR5P). DERA enzymes have been found to accept also other types of aldehydes as their donor, and in particular as acceptor molecules. Consequently, DERA enzymes can be applied in C–C bond formation reactions to produce novel compounds, thus offering a versatile biocatalytic alternative for synthesis. DERA enzymes, found in all kingdoms of life, share a common TIM barrel fold despite the low overall sequence identity. The catalytic mechanism is well-studied and involves formation of a covalent enzyme-substrate intermediate. A number of protein engineering studies to optimize substrate specificity, enzyme efficiency, and stability of DERA aldolases have been published. These have employed various engineering strategies including structure-based design, directed evolution, and recently also machine learning–guided protein engineering. For application purposes, enzyme immobilization and usage of whole cell catalysis are preferred methods as they improve the overall performance of the biocatalytic processes, including often also the stability of the enzyme. Besides single-step enzymatic reactions, DERA aldolases have also been applied in multi-enzyme cascade reactions both in vitro and in vivo. The DERA-based applications range from synthesis of commodity chemicals and flavours to more complicated and high-value pharmaceutical compounds. Key points • DERA aldolases are versatile biocatalysts able to make new C–C bonds. • Synthetic utility of DERAs has been improved by protein engineering approaches. • Computational methods are expected to speed up the future DERA engineering efforts. Graphical abstract

Unlocking Asymmetric Michael Additions in an Archetypical Class I Aldolase by Directed Evolution

“…Natural aldolases are regarded as highly specific for their nucleophilic aldol donor and are restricted by the range of reactions catalyzed. Even though the substrate scope, activity, and stability − , of the archetypical class I aldolase DERA have been successfully altered to enlarge its usefulness for diverse aldol reactions, examples of other mechanistically related carboligation reactions catalyzed by DERA are unknown. In the current study, we demonstrate that the active site of DERA from E.…”

“…Natural aldolases are regarded as highly specific for their nucleophilic aldol donor 7 and are restricted by the range of reactions catalyzed. Even though the substrate scope, activity, and stability [8][9][10][11][12]39 of the archetypical class I aldolase DERA have been successfully altered to enlarge its usefulness for diverse aldol reactions, examples of other mechanistically related carboligation reactions catalyzed by DERA are unknown. In the current study, we demonstrate that the active site of DERA from E. coli can give rise to synthetically useful catalytic promiscuity, supporting asymmetric Michael additions of nitromethane to various α,β-unsaturated aldehydes to give γ-nitroaldehydes, important chiral synthons for pharmaceutically active γ-aminobutyric acids.…”

Synthetic Activity of Recombinant Whole Cell Biocatalysts Containing 2‐Deoxy‐D‐ribose‐5‐phosphate Aldolase from Pectobacterium atrosepticum

“…Therefore, in the last decades considerable attention has been paid to the search for new aldolases from a large diversity of microorganisms with enhanced acetaldehyde resistance. [28,29,22] In a previous work we selected DERA from P. atrosepticum (PaDERA) as an efficient wild-type bacterial whole cell biocatalyst for its ability to synthesize DR5P at up to 200 mM of acetaldehyde. [26] In addition, DR5P was further employed in an one-pot multistep enzymatic synthesis to prepare thymidine.…”

“…Following this strategy, different recombinant DERAs have been reported with the aim of increasing their tolerance to high aldehyde concentrations. [22] DERA has been shown to have a low preference for nonphosphorylated substrates. [23] In order to overcome this, De Santis et al, prepared a mutant of E. coli DERA (S238D) with improved activity towards 2-deoxy-D-ribose.…”

“…They modified the enzyme by changing glutamate 78 to lysine, which also provided a significant improvement in both thermostability and aldehyde tolerance. Following this strategy, different recombinant DERAs have been reported with the aim of increasing their tolerance to high aldehyde concentrations [22] …”